Methods for the Identification, Assessment, Prevention, and Treatment of Neurological Disorders and Diseases Using FNDC5

ABSTRACT

The invention provides methods for identifying, assessing, preventing, and treating neurological disorders and diseases using Fndc5 and modulators of Fndc5 expression or activity.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/885,177, filed on 1 Oct. 2013; the entire contents of saidapplications is incorporated herein in its entirety by this reference.

STATEMENT OF RIGHTS

This invention was made with government support under Grants NIH RO1DK31405 and DK90861 awarded by the National Institutes of Health. TheU.S. government has certain rights in the invention. This statement isincluded solely to comply with 37 C.F.R. §401.14(a)(f)(4) and should notbe taken as an assertion or admission that the application disclosesand/or claims only one invention.

BACKGROUND OF THE INVENTION

Exercise, especially endurance exercise, is known to have beneficialeffects on brain health and cognitive function (Cotman et al. (2007)Trends Neurosci. 30, 464-472 and Mattson (2012) Cell Metab. 16,706-722). This improvement in cognitive function with exercise has beenmost prominently observed in the aging population (Colcombe and Kramer(2003) Psych Sci. 14, 125-130). Exercise has also been reported toameliorate outcomes in neurological diseases like depression, epilepsy,stroke, Alzheimer's and Parkinson's Disease (Ahlskog (2011) Neurology77, 288-294; Arida et al. (2008) Sports Med. (Auckland, NZ) 38, 607-615;Buchman et al. (2012) Neurology 78, 1323-1329; Russo-Neustadt et al.(1999) Neuropsychopharm. 21, 679-682; and Zhang et al. (2012)Neuroscience 205, 10-17). The effects of exercise on the brain are mostapparent in the hippocampus and its dentate gyrus, a part of the braininvolved in learning and memory. Specific beneficial effects of exercisein the brain have been reported to include increases in the size of andblood flow to the hippocampus in humans and morphological changes indendrites and dendritic spines, increased synapse plasticity and,importantly, de novo neurogenesis in the dentate gyrus in various mousemodels of exercise (Cotman et al. (2007) Trends Neurosci. 30, 464-472and Mattson (2012) Cell Metab. 16, 706-722). De novo neurogenesis in theadult brain occurs is observed in only two areas; the dentate gyrus ofthe hippocampus is one of them and exercise is one of the few knownstimuli of this de novo neurogenesis (Kobilo et al. (2011) Learning Mem.(Cold Spring Harbor, N.Y.) 18, 605-609).

One important molecular mediator for these beneficial responses in thebrain to exercise is the induction of neurotrophins/growth factors, mostnotably brain-derived neurotrophic factor (BDNF). In animal models, BDNFis induced in various regions of the brain with exercise and mostrobustly in the hippocampus (Cotman et al. (2007) Trends Neurosci. 30,464-472). BDNF promotes many aspects of brain development includingneuronal cell survival, differentiation, migration, dendriticarborization, synaptogenesis and plasticity (Greenberg et al. (2009) J.Neurosci. 29, 12764-12767 and Park and Poo (2013) Nat. Rev. Neurosci.14, 7-23). In addition, BDNF is essential for synaptic plasticity,hippocampal function and learning (Kuípers et al. (2006) Curr. Opin.Drug Disc. Dev. 9, 580-586). Highlighting the relevance of BDNF inhuman, individuals carrying the Val66Met mutation in the BDNF gene,exhibit decreased secretion of BDNF, display a decreased volume ofspecific brain regions, deficits in episodic memory function as well asincreased anxiety and depression (Egan et al. (2003) Cell 112, 257-269and Haríri et al. (2003) J. Neurosci. 23, 6690-6694). Blocking BDNFsignaling with anti-TrkB antibodies attenuates the exercise-inducedimprovement of acquisition and retention in a spatial learning task, aswell as the exercise-induced expression of synaptic proteins (Vaynman etal. (2004) Eur. J. Neurosci. 20, 2580-2590 and Vaynman et al. (2006)Brain Res. 1070, 124-130). However, the underlying mechanism whichinduces BDNF in exercise remains to be determined.

PGC-1α is induced in skeletal muscle with exercise and is a majormediator of the beneficial effects of exercise in this tissue (Finck andKelly (2006) J. Clin. Invest. 116, 615-622). PGC-1α was initiallydiscovered as a transcriptional co-activator of mitochondrial biogenesisand oxidative metabolism in brown fat (Puigserver et al. (1998) Cell 92,829-839 and Spiegelman (2007) Novartis Foundation Sympos. 287, 60-69).Subsequent work has demonstrated an important role of PGC-1α in thebrain. Lack of PGC-1α in the brain is associated with neurodegeneration(Lin et al. (2004) Cell 119, 121-135 and Ma et al. (2010) J. Biol. Chem.285, 39087-39095), as well as GABAergic dysfunction and a deficiency inneuronal parvalbumin expression (Lucas et al. (2010) J. Neurosci. 30,7227-7235). PGC-1α has been shown to be neuroprotective in the MPTPmouse model of Parkinson's disease (St-Pierre et al. (2006) Cell 127,397-408). It also negatively regulates extrasynaptic NMDA(N-methyl-D-aspartate) receptor activity and thereby reducesexcitotoxicity in rat cortical neurons (Puddifoot et al. (2012) J.Neurosci. 32, 6995-7000). In addition, the involvement of PGC-1α in theformation and maintenance of neuronal dendritic spines has been reportedby Cheng et al. (2012) Nature Comm. 3, 1250 and long-term forcedtreadmill running over 12 weeks increases Pgc1α expression in variousareas of the brain (Steiner et al. (2011) J. Appl. Physiol, 111,1066-1071).

It has been determined that a PGC-1α-dependent myokine, FNDC5, iscleaved and secreted from muscle during exercise and induces some majormetabolic benefits of exercise (Bostrom et al. (2012) Nature 481,463-468). FNDC5 is a glycosylated type I membrane protein and isreleased into the circulation after proteolytic cleavage. The secretedform of FNDC5 contains 112 amino acids and has been named irisin. Irisinacts preferentially on the subcutaneous ‘beige’ fat and causes it to‘brown’ by increasing the expression of UCP-1 and other thermogenicgenes (Bostrom et al. (2012) Nature 481, 463-468 and Wu et al. (2012)Cell 150, 366-376). Clinical studies in humans have confirmed thispositive correlation between increased FNDC5 expression and circulatingirisin with the level of exercise performance (Huh et al. (2012)Metabolism 61, 1725-1738 and Lecker et al. (2012) Circ. Heart Failure 5,812-818).

FNDC5 is also expressed in the brain (Dun et al. (2013) Neurosci. 240,155-162; Ferrer-Martinez et al. (2002) Dev. Dyn. 224, 154-167; andTeufel et al. (2002) Gene 297, 79-83) and in ratpheochromocytoma-derived PC12 cells differentiated into neuron-likecells (Ostadsharif et al. (2011) Diff. Res. Biol. Diversity 81,127-132). Knockdown of FNDC5 in neuronal precursors impaired thedevelopment into mature neurons (Hashemi et al. (2013) Neurosci. 231,296-304) and in vitro application of irisin to mouse H19-7 HNhippocampal cells increased cell proliferation without altering markersof hippocampal neurogenesis (Moon et al. (2013) Metabolism62:1131-1136).

Despite the identification of BDNF and other neuromodulatory (e.g.,neuroprotective) factors as important regulators of neuronal developmentand function, such molecules are unstable, difficult to administer tothe central nervous system, and are non-specific, general moleculeshaving a range of functions on different parts of the central andperipheral nervous systems. A major part of the pathology ofneurodegenerative disease is the progressive destruction and loss ofneurons followed by loss of neurological function. Therapeutic effortshave concentrated on the protection and preservation of the endangeredneurons as well as regeneration of new neurons. While neurotrophins,which are neuroprotective, promote nerve cell growth and survival, andhave been become prime candidates because of their major therapeuticeffects in animal studies, clinical trials using neurotrophinsthemselves as therapeutics have not been successful thus far for thereasons described above. Yet, there is a growing need for suchtherapeutics. Impairment of the nervous system caused byneurodegenerative diseases, such as Alzheimer's disease or Parkinson'sdisease, and the associated disability is devastating for the peoplesuffering from it. In the United States at least one million people arebelieved to suffer from Parkinson's disease and about 60,000 people arenewly diagnosed each year. The annual costs alone for Parkinson'sdisease are estimated at $25 billion per year in the U.S., including thecost of treatment, social security payments and lost income frominability to work.

Accordingly, there is a great need to identify molecular regulators ofsuch neuromodulatory (e.g., neuroprotective) factors having improvedproperties for administration, neuromodulatory specificity, andstability, including the generation of diagnostic, prognostic, andtherapeutic agents to effectively regulate neurological processes insubjects.

SUMMARY OF THE INVENTION

The present invention is based in part on the discovery that Fndc5 andbiologically active fragments thereof are secreted polypeptides whoseexpression is elevated by endurance exercise in the hippocampus andother brain areas; that PGC-1α and FNDC5 regulate BDNF expression in thebrain; and, that FNDC5 promotes survival of neurons and inhibitsneurodegeneration mediated by its effects on BDNF expression. It wasunexpectedly determined that BDNF expression or activity could bemodulated in the central and/or peripheral nervous system in subjects byadministering an Fndc5 or irisin polypeptide, either within the nervoussystem or, surprisingly, systemically in the plasma.

In one aspect, as method of increasing expression of brain-derivedneurotrophic factor (BDNF) by a cell is provided comprising, contactingthe cell with an agent, wherein the agent is selected from the groupconsisting of an Fndc5 polypeptide or fragment thereof, a nucleic acidthat encodes Fndc5 or a fragment thereof, and an enhancer of Fndc5polypeptide and/or nucleic acid expression and/or activity, to therebyincrease the expression of BDNF by the cell. In one embodiment, the stepof contacting occurs in vivo, ex vivo, or in vitro. In anotherembodiment, the cells are neurons (e.g., hippocampal neurons, cerebellarneurons, sciatic nerve neurons, dopaminergic neurons, or substantianigra neurons). In still another embodiment, the method furthercomprises contacting the cell with an additional agent that increasesthe expression of BDNF.

In another aspect, a method for treating or preventing a neurologicaldisease or disorder in a subject is provided comprising the step ofadministering to the subject an agent selected from the group consistingof an Fndc5 polypeptide or fragment thereof, a nucleic acid that encodesFndc5 or a fragment thereof, and an enhancer of Fndc5 polypeptide and/ornucleic acid expression and/or activity, that increases BDNF expressionor activity in the central or peripheral nervous system of the subject,such that the neurological disease or disorder is treated or prevented.In one embodiment, the agent is administered systemically (e.g.,intravenous or subcutaneous administration). In another embodiment, theagent is administered in a pharmaceutically acceptable formulation. Instill another embodiment, the neurological disease or disorder wouldbenefit from decreased neuronal cell death and/or increased neuronalsurvival, optionally wherein the neurological disease or disorder isselected from the group consisting of Alzheimer's disease, Parkinson'sdisease, Huntington's disease, Pick's disease, Kuf's disease, Lewy bodydisease, neurofibrillary tangles, Rosenthal fibers, Mallory's hyaline,senile dementia, myasthenia gravis, Gilles de la Tourette's syndrome,multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS),progressive supranuclear palsy (PSP), epilepsy, Creutzfeldt-Jakobdisease, deafness-dytonia syndrome, Leigh syndrome, Leber hereditaryoptic neuropathy (LHON), parkinsonism, dystonia, motor neuron disease,neuropathy-ataxia and retinitis pimentosa (NARP), maternal inheritedLeigh syndrome (MILS), Friedreich ataxia, hereditary spastic paraplegia,Mohr-Tranebjaerg syndrome, Wilson disease, sporatic Alzheimer's disease,sporadic amyotrophic lateral sclerosis, sporadic Parkinson's disease,autonomic function disorders, hypertension, sleep disorders,neuropsychiatric disorders, depression, schizophrenia, schizoaffectivedisorder, korsakoff's psychosis, mania, anxiety disorders, phobicdisorder, learning or memory disorders, amnesia or age-related memoryloss, attention deficit disorder, dysthymic disorder, major depressivedisorder, obsessive-compulsive disorder, psychoactive substance usedisorders, panic disorder, bipolar affective disorder, severe bipolaraffective (mood) disorder (BP-1), migraines, hyperactivity and movementdisorders. In yet another embodiment, the subject is a human.

In still another aspect, a method for assessing whether a subject isafflicted with a neurological disease or disorder or has a risk ofdeveloping a neurological disease or disorder is provided comprising thesteps of detecting the expression of the Fndc5 gene or the expression oractivity of Fndc5 polypeptide in a sample of a subject, wherein adecrease in the expression of the Fndc5 gene or a decrease in theexpression or activity of the Fndc5 polypeptide compared to a controlindicates the presence of a neurological disease or disorder or the riskof developing a neurological disease or disorder in the subject. In oneembodiment, the sample is selected from the group consisting of wholeblood, serum, plasma, saliva, cerebrospinal fluid, spinal fluid, andneural tissue. In another embodiment, the expression of the Fndc5polypeptide or protein thereof is detected using a reagent whichspecifically binds with the protein (e.g., an antibody, an antibodyderivative, and an antibody fragment). In still another embodiment, theexpression of the Fndc5 gene is assessed by detecting the presence inthe sample of a transcribed polynucleotide or portion thereof (e.g., anmRNA or a cDNA). In yet another embodiment, the step of detectingfurther comprises amplifying the transcribed polynucleotide. In anotherembodiment, the level of expression of the marker in the sample isassessed by detecting the presence in the sample of a transcribedpolynucleotide which anneals with Fndc5 or anneals with a portion of anFndc5 polynucleotide under stringent hybridization conditions. In stillanother embodiment, the neurological disease or disorder would benefitfrom decreased neuronal cell death and/or increased neuronal survival,optionally wherein the neurological disease or disorder is selected fromthe group consisting of Alzheimer's disease, Parkinson's disease,Huntington's disease, Pick's disease, Kuf's disease, Lewy body disease,neurofibrillary tangles, Rosenthal fibers, Mallory's hyaline, seniledementia, myasthenia gravis, Gilles de la Tourette's syndrome, multiplesclerosis (MS), amyotrophic lateral sclerosis (ALS), progressivesupranuclear palsy (PSP), epilepsy, Creutzfeldt-Jakob disease,deafness-dytonia syndrome, Leigh syndrome, Leber hereditary opticneuropathy (LHON), parkinsonism, dystonia, motor neuron disease,neuropathy-ataxia and retinitis pimentosa (NARP), maternal inheritedLeigh syndrome (MILS), Friedreich ataxia, hereditary spastic paraplegia,Mohr-Tranebjaerg syndrome, Wilson disease, sporatic Alzheimer's disease,sporadic amyotrophic lateral sclerosis, sporadic Parkinson's disease,autonomic function disorders, hypertension, sleep disorders,neuropsychiatric disorders, depression, schizophrenia, schizoaffectivedisorder, korsakoff's psychosis, mania, anxiety disorders, phobicdisorder, learning or memory disorders, amnesia or age-related memoryloss, attention deficit disorder, dysthymic disorder, major depressivedisorder, obsessive-compulsive disorder, psychoactive substance usedisorders, panic disorder, bipolar affective disorder, severe bipolaraffective (mood) disorder (BP-1), migraines, hyperactivity and movementdisorders. In yet another embodiment, the subject is a human.

In yet another aspect, a method for assessing the efficacy of an agentthat treats or prevents a neurological disease or disorder in a subjectis provided comprising: a) detecting in a subject sample at a firstpoint in time BDNF polypeptide or nucleic acid expression and/oractivity in the central and/or peripheral nervous system; b) repeatingstep a) during at least one subsequent point in time afteradministration of the agent, wherein the agent is selected from thegroup consisting of an Fndc5 polypeptide or fragment thereof, a nucleicacid that encodes Fndc5 or a fragment thereof, and an enhancer of Fndc5polypeptide and/or nucleic acid expression and/or activity; and c)comparing the expression and/or activity detected in steps a) and b),wherein a significantly increased BDNF polypeptide or nucleic acidexpression and/or activity in the first subject sample relative to atleast one subsequent subject sample, indicates that the agent treats orprevents the neurological disease or disorder in the subject. In oneembodiment, the first and/or at least one subsequent sample is selectedfrom the group consisting of whole blood, serum, plasma, saliva,cerebrospinal fluid, spinal fluid, and neural tissue. In anotherembodiment, the subject has undergone treatment, completed treatment,and/or is in remission for the neurological disease or disorder inbetween the first point in time and the subsequent point in time. Instill another embodiment, the first and/or at least one subsequentsample is selected from the group consisting of ex vivo and in vivosamples. In yet another embodiment, the first and/or at least onesubsequent sample is obtained from an animal model of the neurologicaldisease or disorder. In another embodiment, the first and/or at leastone subsequent sample is a portion of a single sample or pooled samplesobtained from the subject. In still another embodiment, the expressionof the BDNF polypeptide is detected using a reagent which specificallybinds with the protein (e.g., an antibody, an antibody derivative, andan antibody fragment). In yet another embodiment, the expression of theBDNF nucleic acid is assessed by detecting the presence in the sample ofa transcribed polynucleotide or portion thereof (e.g., an mRNA or acDNA). In another embodiment, the step of detecting further comprisesamplifying the transcribed polynucleotide. In still another embodiment,the level of expression of the marker in the sample is assessed bydetecting the presence in the sample of a transcribed polynucleotidewhich anneals with BDNF or anneals with a portion of a BDNFpolynucleotide under stringent hybridization conditions.

In another aspect, a cell-based assay for screening for agents thatmodulate the ability of the cell to increase BDNF expression is providedcomprising contacting the cell with a test agent selected from the groupconsisting of an Fndc5 polypeptide or fragment thereof, a nucleic acidthat encodes Fndc5 or a fragment thereof, and an enhancer of Fndc5polypeptide and/or nucleic acid expression and/or activity, anddetermining the ability of the test agent to increase BDNF expression bythe cell. In one embodiment, the step of contacting occurs in vivo, exvivo, or in vitro. In another embodiment, the cells are neurons (e.g.,hippocampal neurons, cerebellar neurons, sciatic nerve neurons,dopaminergic neurons, or substantia nigra neurons).

Further provided are embodiments that can be applied to any compositionor method of the present invention described herein. For example, in oneembodiment, the Fndc5 polypeptide is selected from the group ofpolypeptides consisting of: a) a polypeptide encoded by a nucleic acidmolecule comprising a nucleotide sequence encoding a fragment of theFNDC5 polypeptide of SEQ ID NO: 2, wherein said fragment lacks theC-terminal domain sequence of said FNDC5 polypeptide, and wherein saidpolypeptide has one or more of the biological activities of said FNCD5polypeptide; b) an isolated polypeptide encoded by a nucleic acidmolecule comprising a nucleotide sequence encoding an amino acidsequence that is at least 70% identical to the amino acid sequence ofresidues 73-140 of the FNDC5 polypeptide of SEQ ID NO:2, wherein saidpolypeptide does not encode the C-terminal domain sequence of said FNDC5polypeptide, and wherein said polypeptide has one or more of thebiological activities of said FNCD5 polypeptide; c) a polypeptide whichis a fragment of the FNDC5 polypeptide SEQ ID NO: 2, which fragment isoptionally fused to one or more heterologous polypeptides at itsN-terminus and/or C-terminus, wherein said fragment consists of asequence of amino acids in between residues 1 and 150 of SEQ ID NO: 2,and wherein said fragment has one or more of the biological activitiesof said FNCD5 polypeptide; and d) a polypeptide which is a fragment ofthe FNDC5 polypeptide of SEQ ID NO: 4, 6 or 8, wherein said fragment isoptionally fused to one or more heterologous polypeptides at itsN-terminus and/or C-terminus, and wherein said fragment has one or moreof the biological activities of said FNCD5 polypeptide. In anotherembodiment, the Fndc5 polypeptide is selected from the group ofpolypeptides consisting of: a) an isolated polypeptide fragment of anFndc5 protein comprising at least one fibronectin domain and is notfull-length Fndc5; b) an isolated polypeptide fragment of an Fndc5protein comprising at least one fibronectin domain and which lacks oneor more functional domain(s) selected from the group consisting ofsignal peptide, hydrophobic, and C-terminal domains; c) an isolatedpolypeptide comprising an amino acid sequence that is at least 70%identity to the amino acid sequence comprising residues 73-140 of SEQ IDNO:2, residues 30-140 of SEQ ID NO:2 or residues 29-140 of SEQ ID NO:2and which lacks one or more functional domain(s) of an Fndc5 proteinselected from the group consisting of signal peptide, hydrophobic, andC-terminal domains; d) an isolated polypeptide comprising an amino acidsequence that is at least 70% identity to the amino acid sequencecomprising residues 73-140 of SEQ ID NO:2, residues 30-140 or SEQ IDNO:2 or residues 29-140 or SEQ ID NO:2 and which is less than 195 aminoacids in length; e) an isolated polypeptide consisting essentially of anamino acid sequence that is at least 70% identity to the amino acidsequence comprising residues 73-140 of SEQ ID NO:2, residues 30-140 ofSEQ ID NO:2 or residues 29-140 of SEQ ID NO:2; f) an isolatedpolypeptide fragment of SEQ ID NO:2 comprising residues 73-140 of SEQ IDNO:2, residues 30-140 of SEQ ID NO:2 or residues 29-140 of SEQ ID NO:2and which is not full-length; g) an isolated polypeptide fragment of SEQID NO:2 consisting essentially of residues 73-140 of SEQ ID NO:2,residues 30-140 of SEQ ID NO:2 or residues 29-140 of SEQ ID NO:2; h) anisolated polypeptide which is encoded by a nucleic acid moleculecomprising a nucleotide sequence encoding at least one fibronectindomain of an Fndc5 protein and does not encode full-length Fndc5; i) anisolated polypeptide fragment of an Fndc5 protein which is encoded by anucleic acid molecule comprising a nucleotide sequence encoding at leastone fibronectin domain and which does not encode one or more functionaldomain(s) selected from the group consisting of signal peptide,hydrophobic, and C-terminal domains; j) an isolated polypeptide which isencoded by a nucleic acid molecule comprising a nucleotide sequenceencoding an ammo acid sequence that is at least 70% identical to theamino acid sequence of residues 73-140 of SEQ ID NO:2, residues 30-140of SEQ ID NO:2 or residues 29-140 of SEQ ID NO:2 and which does notencode one or more functional domain(s) of an Fndc5 protein selectedfrom the group consisting of signal peptide, hydrophobic, and C-terminaldomains; k) an isolated polypeptide which is encoded by a nucleic acidmolecule comprising a nucleotide sequence encoding an amino acidsequence that is at least 70% identical to the amino acid sequence ofresidues 73-140 of SEQ ID NO:2, residues 30-140 of SEQ ID NO:2 orresidues 29-140 of SEQ ID NO:2 and which is less than 630 nucleotides inlength; l) an isolated polypeptide which is encoded by a nucleic acidmolecule consisting essentially of a nucleotide sequence encoding anamino acid sequence having at least 70% identity to the amino acidsequence of residues 73-140 of SEQ ID NO:2, residues 30-140 of SEQ IDNO:2 or residues 29-140 of SEQ ID NO:2; m) an isolated polypeptide whichis encoded by a nucleic acid molecule comprising a nucleotide sequenceencoding an amino acid sequence that is at least 70% identical to theamino acid sequence of residues 73-140 of SEQ ID NO:2, residues 30-140of SEQ ID NO:2 or residues 29-140 of SEQ ID NO:2 and which does notencode the full-length amino acid sequence of SEQ ID NO:2; n) anisolated polypeptide which is encoded by a nucleic acid moleculecomprising a nucleotide sequence encoding the amino acid sequence ofresidues 73-140 of SEQ NO:2, residues 30-140 of SEQ ID NO:2 or residues29-140 of SEQ ID NO:2 and which does not encode the full-length aminoacid sequence of SEQ ID NO:2; o) an isolated polypeptide which isencoded by a nucleic acid molecule consisting essentially of anucleotide sequence encoding the amino acid sequence of residues 73-140of SEQ ID NO:2, residues 30-140 of SEQ ID NO:2 or residues 29-140 of SEQID NO:2; p) an isolated polypeptide which is encoded by a nucleic acidmolecule comprising a nucleotide sequence which is at least 70%identical to the nucleotide sequence of nucleotides 217-420 of SEQ IDNO:1, SEQ ID NO:15, nucleotides 88-420 of SEQ ID NO:1, or nucleotides85-420 of SEQ ID NO:1 and which does not encode one or more functionaldomain(s) of an Fndc5 protein selected from the group consisting ofsignal peptide, hydrophobic, and C-terminal domains; and q) an isolatedpolypeptide which is encoded by a nucleic acid molecule consistingessentially of a nucleotide sequence which is at least 70% identical tothe nucleotide sequence of nucleotides 217-420 of SEQ ID NO:1, SEQ IDNO:15, nucleotides 88-420 of SEQ ID NO:1, or nucleotides 85-420 of SEQID NO:1.

In still another embodiment, the one or more of the biologicalactivities of FNDC5 polypeptide is selected from the group consistingof 1) increasing BDNF expression in the central and/or peripheralnervous system; 2) increasing activity-induced immediate-early geneexpression in neurons; 3) increasing neuronal survival; 4) decreasingneurological lesion formation; 5) increasing neurite outgrowth; 6)increasing synaptogenesis; 7) increasing synaptic plasticity; 8)decreasing neuronal mitochondrial dysfunction; 9) increasing dendriticarborization; 10) increasing neuronal differentiation; and 11)increasing neuronal migration. In yet another embodiment, the fragmentor encoded amino acid sequence is more than 65 amino acids in lengthand/or less than 135 amino acids in length. In another embodiment, thepolypeptide is between 70 and 125 amino acids in length or is less than195 amino acids in length. In still another embodiment, the polypeptideis a fragment of SEQ ID NO: 2 which consists of about amino acids 30 to140 or 73-140 SEQ ID NO: 2, wherein said fragment is optionally fused toone or more heterologous polypeptides at its N-terminus and/orC-terminus. In yet another embodiment, the polypeptide comprises afibronectin domain. In another embodiment, the polypeptide isglycosylated or pegylated, optionally wherein at least one glycosylatedamino acid residue corresponds to asparagine at position 36 and/or theasparagine at position 81 of SEQ ID NO:2. In still another embodiment,the polypeptide comprises an amino acid sequence that is heterologous tosaid FNDC5 polypeptide (e.g., an Fc domain, an IgG1 Fc domain, an IgG2Fc domain, an IgG3 Fc domain, and IgG4 Fc domain, a dimerization domain,an oligomorization domain, an agent that promotes plasma solubility,albumin, a signal peptide, a peptide tag, a 6-His tag, a thioredoxintag, a hemaglutinin tag, a GST tag, or an OmpA signal sequence tag). Inyet another embodiment, the polypeptide can cross the blood-brainbarrier. In another embodiment, the Fndc5 nucleic acid encodes apolypeptide of claim 38-47. In still another embodiment, the Fndc5nucleic acid is selected from the group consisting of: a) a nucleic acidmolecule comprising a nucleotide sequence encoding a fragment of theFNDC5 polypeptide SEQ ID NO: 2, wherein said fragment lacks theC-terminal domain sequence of said FNDC5 polypeptide, and wherein saidfragment has one or more of the biological activities of said FNCD5polypeptide; b) a nucleic acid molecule which encodes a polypeptidecomprising an amino acid sequence having at least 70% identity to theamino acid sequence of residues 73-140 of the FNDC5 polypeptide of SEQID NO:2, wherein said polypeptide does not encode the C-terminal domainsequence of said FNDC5 polypeptide, and wherein said polypeptide has oneor more of the biological activities of said FNCD5 polypeptide; and c) anucleic acid molecule which encodes a fibronectin domain of the FNCD5polypeptide of SEQ ID NO: 2 but which does not encode the full lengthsequence of SEQ ID NO: 2. In yet another embodiment, the Fndc5 nucleicacid is selected from the group consisting of: a) an isolated nucleicacid molecule which encodes at least one fibronectin domain of an Fndc5protein and which does not encode full-length Fndc5; b) an isolatednucleic acid molecule which encodes at least one fibronectin domain ofan Fndc5 protein and which does not encode one or more functionaldomain(s) of an Fndc5 protein selected from the group consisting ofsignal peptide, hydrophobic, and C-terminal domains; c) an isolatednucleic acid molecule which encodes a polypeptide comprising an aminoacid sequence having at least 70% identity to the 88-amino acid sequenceof residues 73-140 of SEQ ID NO:2, residues 30-140 of SEQ ID NO:2 orresidues 29-140 of SEQ ID NO:2 and which does not encode one or morefunctional domain(s) of an Fndc5 protein selected from the groupconsisting of signal peptide, hydrophobic, and C-terminal domains; d) anisolated nucleic acid molecule which encodes a polypeptide comprising anamino acid sequence having at least 70% identity to the amino acidsequence of residues 73-140 of SEQ ID NO:2, residues 30-140 of SEQ IDNO:2 or residues 29-140 of SEQ NO:2 and which is less than 630nucleotides in length; e) an isolated nucleic acid molecule whichencodes a polypeptide consisting essentially of an amino acid sequencehaving at least 70% identity to the amino acid sequence of residues73-140 of SEQ ID NO:2, residues 30-140 of SEQ ID NO:2 or residues 29-140of SEQ ID NO:2; f) an isolated nucleic acid molecule which encodes apolypeptide comprising an amino acid sequence having at least 70%identity to the amino acid sequence of residues 73-140 of SEQ ID NO:2,residues 30-140 of SEQ ID NO:2 or residues 29-140 of SEQ ID NO:2 andwhich does not encode the full-length amino acid sequence SEQ ID NO:2;g) an isolated nucleic acid molecule which encodes a polypeptidecomprising the amino acid sequence of residues 73-140 of SEQ ID NO:2,residues 30-140 of SEQ ID NO:2 or residues 29-140 of SEQ ID NO:2 andwhich does not encode the full-length amino acid sequence of SEQ IDNO:2: b) an isolated nucleic acid molecule which encodes a polypeptideconsisting essentially of the amino acid sequence of residues 73-140 ofSEQ ID NO:2, residues 30-140 of SEQ ID NO:2 or residues 29-140 of SEQ IDNO:2; i) an isolated nucleic acid molecule comprising a nucleotidesequence which is at least 70% identical to the nucleotide sequence ofnucleotides 217-420 of SEQ ID NO:1, SEQ ID NO:15, nucleotides 88-420 SEQID NO:1, or nucleotides 85-420 of SEQ ID NO:1 and which does not encodeone or more functional domain(s) of an Fndc5 protein selected from thegroup consisting of signal peptide, hydrophobic, and C-terminal domains;and j) an isolated nucleic acid molecule consisting essentially of anucleotide sequence which is at least 70% identical to the nucleotidesequence of nucleotides 217-420 of SEQ ID NO:1, SEQ ID NO:15,nucleotides 88-420 of SEQ ID NO:1, or nucleotides 85-420 of SEQ ID NO:1.

BRIEF DESCRIPTION OF FIGURES

FIGS. 1A-1E shows that endurance exercise induces hippocampal Fndc5 geneexpression. FIGS. 1A-1E show the results of male six week old C57/B16wild type mice that were individually housed in cages with access to arunning-wheel (free wheel-running) or without (sedentary). Mice wereexercised for 30 days and sacrificed approximately 10 h after their lastbout of exercise. The quadriceps muscle (quadriceps) was harvested. Thebrain was retrieved and the hippocampus was dissected out. mRNA wasprepared and gene expression was assessed by qPCR. Data are shown asmRNA levels relative to Rsp18 expression, expressed as mean±SEM. *P<0.05compared to sedentary control group.

FIG. 2 shows an adaptive endurance exercise response in quadricepsmuscle using qPCR analyses from mice treated as described in FIG. 1.

FIGS. 3A-3C shows that Fndc5 gene expression correlates with Pgc1aexpression levels in various tissues and developmental stages. FIG. 3Ashows the results of the indicated tissues harvested from male 13 weekold C57/B16 wild type mice. mRNA was prepared and gene expression wasassessed by qPCR. Data are shown as mRNA levels relative to Rsp18expression, expressed as mean±SEM. Quad=quadriceps muscle,Gastroc=gastrocnemius muscle, Sp. Cord=spinal cord, ingWAT=inguinalwhite adipose tissue, epiWAT=epididymal white adipose tissue,iBAT=interscapular brown adipose tissue. FIG. 3B shows the results ofbrains harvested from C57/B16 wild type mice at the indicated postnatal(P) time points. mRNA was prepared and gene expression was assessed byqPCR. Data are shown as mRNA levels relative to Rsp18 expression,expressed as mean±SEM. FIG. 3C shows the results of primary corticalneurons isolated from C57/B16 wild type E17 embryos and cultured invitro. At the indicated days in vitro (DIV) mRNA was prepared and geneexpression was assessed by qPCR. Data are shown as mRNA levels relativeto Rsp18 expression, expressed as mean±SEM. *P<0.05 compared to DIV 1control group.

FIGS. 4A-4E show that neuronal Fndc5 gene expression is regulated byPGC-1α. FIG. 4A shows the results of primary cortical neurons at DIV 7treated with either forskolin (10 μM), a stimulator intracellular cAMPlevels, or vehicle for overnight. mRNA was prepared and gene expressionwas assessed by qPCR. Data are shown as mRNA levels relative to Rsp18expression, expressed as mean±SEM. *P<0.05 compared to vehicle onlygroup. FIG. 4B shows the results of primary cortical neurons at DIV 7treated with nifedipine (5 μM), a L-type calcium channel blocker, orvehicle for overnight. mRNA was prepared and gene expression wasassessed by qPCR. Data are shown as mRNA levels relative to Rsp18expression, expressed as mean±SEM. *P<0.05 compared to vehicle onlygroup. FIG. 4C shows the results of primary cortical neurons at DIV 7transduced with either PGC-1α or GFP adenovirus. Forty-eight hours latermRNA was prepared and gene expression was assessed by qPCR. Data areshown as mRNA levels relative to Rsp18 expression, expressed asmean±SEM. *P<0.05 compared to corresponding GFP expressing controlgroup. FIG. 4D shows the results of primary cortical neurons at DIV 5transduced with lentivirus carrying the specified shRNA hairpins againstPgc1a or luciferase (Luc) as control. Four days later mRNA was preparedand gene expression was assessed by qPCR. Data are shown as mRNA levelsrelative to Rsp18 expression, expressed as mean±SEM. *P<0.05 compared tocorresponding shLuc expressing control group. FIG. 4E shows the resultsof cortices harvested from either male five months old Pgc1a KO(Pgc1−/−) or wild type mice (Pgc1a+/+). mRNA was prepared and geneexpression was assessed by qPCR. Data are shown as mRNA levels relativeto Rsp18 expression, expressed as mean±SEM. *P<0.05 compared to wildtype control group.

FIG. 5 shows the results of primary cortical neurons at DIV 6 transducedwith either PGC-1α or GFP adenovirus. Forty-eight hours later, wholecell lysates were harvested and analyzed by immunoblotting. *=unspecificband. Intensity of unspecific bands and Ponceau staining were used toassess equal loading.

FIGS. 6A-6D show that ERRα is a key interacting transcription factorwith PGC-1α for regulating Fndc5 gene expression in neurons. FIG. 6Ashows the results of primary cortical neurons at DIV 7 transduced witheither PGC-1α or GFP adenovirus. Forty-eight hours later mRNA wasprepared and gene expression was assessed by qPCR. Data are shown asmRNA levels relative to Rsp18 expression, expressed as mean±SEM. *P<0.05compared to corresponding GFP expressing control group. FIG. 6B showsthe results of primary cortical neurons at DIV 7 treated with either XCT790 (1 μM), a selective inverse ERRα agonist, DY131 (1 μM), a selectiveERRβ and ERRγ agonist, or vehicle for overnight. mRNA was prepared andgene expression was assessed by qPCR. Data are shown as mRNA levelsrelative to Rsp18 expression, expressed as mean±SEM, *P<0.05 compared tovehicle only group. FIG. 6C shows the results of primary corticalneurons at DIV 4 transduced with lentivirus carrying shRNA hairpinsagainst either Erra or luciferase (Luc) as control. Three days latercells were transduced with either PGC-1α or GFP adenovirus. Forty-eighthours later mRNA was prepared and gene expression was assessed by qPCR.Data are shown as mRNA levels relative to Rsp18 expression, expressed asmean±SEM. *P<0.05 compared to corresponding shLuc expressing controlgroup. *P<0.05 compared to corresponding GFP expressing control group.FIG. 6D shows the results of analyzing the murine Fndc5 promoter forERREs. The murine Fndc5 gene and 6 kb of its upstream promoter weresearched for the canonical ERRE: TGA CCTT. Genomic coordinates are givenaccording to the assembly mm9 from the UCSC Genome Browser. The bottomdiagram indicates the degree of mammalian conservation across thegenomic locus. The presented motif was modified from an online toolavailable on the World Wide Web at factorbook.org (Wang et al. (2012)Genome Res. 22, 1798-1812).

FIGS. 7A-7C provide additional data demonstrating that ERRα is a keyinteracting transcription factor with PGC-1α for regulating Fndc5 geneexpression in neurons. FIG. 7A shows the results of primary corticalneurons at DIV 7 transduced with either PGC-1α or GFP adenovirus.Forty-eight hours later mRNA was prepared and gene expression wasassessed by qPCR. Data are shown as mRNA levels relative Rsp18expression, expressed as mean±SEM. *P<0.05 compared to corresponding GFPexpressing control group. FIG. 7B shows the results of primary corticalneurons at DIV 7 treated with either GW7647 (1 μM), a potent and highlyselective PPARα agonist, GW0742 (1 μM), a potent and highly selectivePPARδ or vehicle for overnight. mRNA was prepared and gene expressionwas assessed by qPCR. Data are shown as mRNA levels relative to Rsp18expression, expressed as mean±SEM. *P<0.05 compared to vehicle onlygroup. FIG. 7C shows the results of primary cortical neurons at DIV 4transduced with lentivirus carrying shRNA hairpins against either Erraluciferase (Luc) as control. Three days later cells were transduced witheither PGC-1α or GFP adenovirus. Forty-eight hours later mRNA wasprepared and gene expression was assessed by qPCR. Data are shown asmRNA levels relative to Rsp18 expression, expressed as mean±SEM. *P<0.05compared to corresponding shLuc expressing control group. $P<0.5compared to corresponding GFP expressing control group.

FIGS. 8A-8H shows that FNDC5 regulates Bdnf gene expression in acell-autonomous manner and recombinant BDNF decreases Fndc5 geneexpression as part of negative feedback loop. FIG. 8A shows the resultsof primary cortical neurons at DIV 6 transduced with either FNDC5 or GFPadenovirus. Forty-eight hours later, cells were washed with PBS andplain neurobasal was added. Whole cell lysates and conditioned mediawere harvested the next day. Conditioned media was concentrated anddeglycosylated. Samples were analyzed by immunoblotting. Intensity ofunspecific bands and Ponceau staining were used to assess equal loading.deglyc.=deglycosylation. FIG. 8B shows the results of primary corticalneurons at DIV 7 transduced with either FNDC5 or GFP adenovirus.Forty-eight hours later mRNA was prepared and gene expression wasassessed by qPCR. Data are shown as mRNA levels relative to Rsp18expression, expressed as mean±SEM. *P<0.05 compared to corresponding GFPexpressing control group. FIG. 8C shows the results of primary corticalneurons at DIV 5 transduced with lentivirus carrying the specified shRNAhairpins against Fndc5 or luciferase (Luc) as control. Four days latermRNA was prepared and gene expression was assessed by qPCR. Data areshown as mRNA levels relative to Rsp18 expression, expressed asmean±SEM. *P<0.05 compared to corresponding shLuc expressing controlgroup. FIG. 8D shows the results of primary cortical neurons at DIV 7transduced with either FNDC5 or GFP adenovirus. Three days later cellviability was assessed using the CellTiter-Glo® Luminescent CellViability Assay (Promega). Data are expressed as mean±SEM and shown asfold compared to GFP expressing control group. *P<0.05 compared to theGFP expressing control group. AU=arbitrary unit. FIG. 8E shows theresults of primary cortical neurons at DIV 5 transduced with lentiviruscarrying the specified shRNA hairpins against Fndc5 or luciferase (Luc)as control. Three days later cell viability was assessed using theCellTiter-Glo® Luminescent Cell Viability Assay (Promega). Data areexpressed as mean±SEM and shown as fold compared to the shLuc expressingcontrol group. *P<0.05 compared to the shLuc expressing control group.AU=arbitrary unit. FIG. 8F shows the results of primary cortical neuronsat DIV 7 stimulated with human recombinant BDNF at the indicatedconcentrations or vehicle for overnight. mRNA was prepared and geneexpression was assessed by qPCR. Data are shown as mRNA levels relativeto Rsp18 expression, expressed as mean±SEM. *P<0.05 compared to vehicleonly group. FIG. 8G shows the results of primary cortical neurons at DIV7 stimulated with the indicated recombinant neurotrophins and growthfactors (100 ng/ml) for overnight. mRNA was prepared and gene expressionwas assessed by qPCR. Data are shown as mRNA levels relative to Rsp18expression, expressed as mean±SEM. *P<0.05 compared to vehicle onlygroup. FIG. 8H shows the results of primary cortical neurons at DIV 6treated either with the TrkB inhibitor K252a (50 nM) or vehicle.Twenty-four hours later human recombinant BDNF (100 ng/ml) or vehiclewas added for overnight stimulation. mRNA was prepared and geneexpression was assessed by qPCR. Data are shown as mRNA levels relativeRsp18 expression, expressed as mean±SEM. *P<0.05 compared to vehicleonly group.

FIGS. 9A-9D shows that peripheral delivery of FNDC5 by adenoviralvectors increases Bdnf expression in the hippocampus. FIGS. 9A-9C showsthe results of five week old male wild-type BALB/c mice injected withGFP- or FNDC5-expressing adenoviral particles intravenously. Animalswere sacrificed seven days later and inguinal/subcutaneous fat pads(WAT=while adipose tissues) (FIG. 9A), hippocampus (FIG. 9B), andforebrain (FIG. 9C) were collected. mRNA was prepared, and geneexpression was assessed by qPCR. Data are shown as mRNA levels relativeto Rsp18 expression, expressed as mean±SEM. *P<0.05 compared to wildtype control group. FIG. 9D shows a model of the hippocampalPGC-1α/FNDC5/BDNF pathway in exercise. Endurance exercise increaseshippocampal Fndc5 gene expression through a PGC-1α/Errα transcriptionalcomplex. This elevated Fndc5 gene expression stimulates in turn Bdnfgene expression. BDNF is the master regulator of nerve cell survival,differentiation and plasticity in the brain. This will lead to improvedcognitive function, learning and memory, which are known beneficialeffects of exercise on the brain.

FIG. 10 provides additional data showing that peripheral delivery ofFNDC5 by adenoviral vectors increases Bdnf expression in thehippocampus. Five week old male wild-type BALB/c mice were injected withGFP- or FNDC5-expressing adenoviral particles intravenously. Animalswere sacrificed seven days later. Plasma samples were collected,depleted from albumin/IgG, deglycosylated, and subjected to WB analysisas shown.

FIGS. 11A-11F show that the PGC-1α/FNDC5/BDNF pathway functions inprimary hippocampal neurons. FIG. 11A shows the results of primaryhippocampal neurons isolated from C57/B16 wild type E17 embryos andcultured in vitro. At the indicated days in vitro (DIV) mRNA wasprepared and gene expression was assessed by qPCR. Data are shown asmRNA levels relative to Rsp18 expression, expressed as mean±SEM. *P<0.05compared to DIV 1 control group. FIG. 11B shows the results of primaryhippocampal neurons at DIV 7 transduced with either PGC-1α or GFPadenovirus. Forty-eight hours later mRNA was prepared and geneexpression was assessed by qPCR. Data are shown as mRNA levels relativeto Rsp18 expression, expressed as mean±SEM. *P<0.05 compared tocorresponding GFP expressing control group. FIG. 11C shows the resultsof primary hippocampal neurons at DIV 5 transduced with lentiviruscarrying the specified shRNA hairpin against Pgc1a or luciferase (Luc)as control. Four days later mRNA was prepared and gene expression wasassessed by qPCR. Data are shown as mRNA levels relative to Rsp18expression, expressed as mean±SEM. *P<0.05 compared to correspondingshLuc expressing control group. FIG. 11D shows the results of primaryhippocampal neurons at DIV 5 and 6 stimulated with recombinant irisin (1ug/ml). mRNA was prepared twenty-four hours and gene expression wasassessed by qPCR. Data are shown as mRNA levels relative to Rsp18expression, expressed as mean±SEM. *P<0.05 compared to vehicle onlygroup. FIG. 11A shows the results of primary hippocampal neurons at DIV5 transduced with lentivirus carrying the specified shRNA hairpinsagainst Fndc5 or luciferase (Luc) as control. Four days later mRNA wasprepared and gene expression was assessed by qPCR. Data are shown asmRNA levels relative to Rsp18 expression, expressed as mean±SEM. *P<0.05compared to corresponding shLuc expressing control group. FIG. 11F showsthe results of primary hippocampal neurons at DIV 7 stimulated withrecombinant BDNF (100 ng/ml) for overnight. mRNA was prepared and geneexpression was assessed by qPCR. Data are shown as mRNA levels relativeto Rsp18 expression, expressed as mean±SEM. *P<0.05 compared to vehicleonly group.

FIG. 12 provides additional data showing that the PGC-1α/FNDC5/BDNFpathway functions in primary hippocampal neurons. Primary hippocampalneurons at DIV 7 were treated with either forskolin (10 μM), astimulator intracellular cAMP levels, or vehicle for overnight. mRNA wasprepared and gene expression was assessed by qPCR. Data are shown asmRNA levels relative to Rsp18 expression, expressed as mean±SEM. *P<0.05compared to vehicle only group.

FIG. 13 shows that a secreted from of FNDC5 is sufficient to increaseBdnf gene expression in neurons release a secreted form of FNDC5 intoculture media. Primary cortical neurons were treated with conditionedmedia (CM, 5× concentrated) from CHO cell lines overexpressing irisinFcor human Fc (hFc) as control. Total RNA for qPCR was harvested the nextday. Results are shown as mean±SEM, n=3 and n=4, respectively. *P<0.05.

FIG. 14 shows that neurons bind irisinFc. PEA-fixed cultured primarycortical neurons were incubated with either irisin-Fc or humanFc ascontrol and binding of irisinFc was detected by a secondary anti-humanFc fluorescent antibody. IrisinFc clearly binds to neurons in culture,especially given that the observed pattern of the binding and the factthat the cultures are highly enriched in neurons (>90%).

FIG. 15 shows that irisin promotes cell survival of primary corticalneurons. Primary cortical neurons were treated with either irisin-Fc orhumanFc as control during in vivo culture for 7 days and cell viabilitywas assessed using the CellGlo™ Assay from Promega as described in theExamples.

FIG. 16 shows that peripheral injections of irisin-Fc increase Bdnf geneexpression in the cerebellum. Wild type C56/B16 mice were injected witheither irisinFc or humanFc (5 mg/kg) i.p. Total RNA for qPCR washarvested 10 d later. Results are shown as mean±SEM, n=6, *P<0.05.

FIG. 17 shows that peripheral injections of irisin-Fc increase Bdnf geneexpression in the sciatic nerve. Wild type C56/B16 mice were injectedwith either irisinFc or humanFc (5 mg/kg) i.p. Total RNA for qPCR washarvested 10 d later. Results are shown as mean±SEM, n=6, *P<0.05.

FIG. 18 shows that Fndc5 expression is reduced by MPTP treatment. Micewere treated with MPTP (4 mg/kg/d), the subtantia nigra was harvested 2days later and gene expression was analyzed by microarray. n=3. Datafrom Phani et al. (2010) Brain Res. 1343:1-13.

FIG. 19 shows that Fndc5 gene expression increases duringdifferentiation of SH-SY5Y neurons. The cells were differentiated withretinoic acid. Gene expression was assessed with qPCR.

FIG. 20 shows that treatment of SH-SY5Y neurons with the neurotoxin,rotenone, reduces Fndc5 gene expression. Gene expression was assessedwith qPCR and the results are consistent with the results shown in FIG.18.

FIG. 21 shows that human embryonic stem cells differentiated into motorneurons (eMN) express Fndc5, with the predicted isoform Fndc5.2 beingthe most abundant. Gene expression was assessed with qPCR.

FIG. 22 shows that irisin-Fc promotes motor neuron differentiation ofeMN (top panel) and increases eMN synapse formation (bottom panel). Geneexpression was assessed with qPCR.

FIGS. 23-26 show that Fndc5 modulates neuronal signaling.

For every figure described herein depicting box plots, the order ofdisplayed boxes from top to bottom in the box plot legend corresponds tothe boxes in the box plot in order from left to right.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based in part on the discovery that Fndc5 oririsin polypeptide, or fragments thereof, can act on neurons of thecentral and/or peripheral nervous system to enhance BDNFexpression/activity and increase neuronal survival and function tothereby prevent or treat undesired neurological disorders.

In order that the present invention may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

As used herein, the term “administering” a substance, such as atherapeutic entity to an animal or cell” is intended to refer todispensing, delivering or applying the substance to the intended target.In terms of the therapeutic agent, the term “administering” is intendedto refer to contacting or dispensing, delivering or applying thetherapeutic agent to an animal by any suitable route for delivery of thetherapeutic agent to the desired location in the animal, includingdelivery by either the parenteral or oral route, intramuscularinjection, subcutaneous/intradermal injection, intravenous injection,buccal administration, transdermal delivery and administration by theintranasal or respiratory tract route.

The term “amino acid” is intended to embrace all molecules, whethernatural or synthetic, which include both an amino functionality and anacid functionality and capable of being included in a polymer ofnaturally-occurring amino acids. Exemplary amino acids includenaturally-occurring amino acids; analogs, derivatives and congenersthereof; amino acid analogs having variant side chains; and allstereoisomers of any of any of the foregoing. The names of the naturalamino acids are abbreviated herein in accordance with therecommendations of IUPAC-IUB.

The term “BDNF” refers to brain-derived neurotrophic factor and is aneurotrophin. The term, “neurotrophins” refers to a class ofstructurally related growth factors that promote neural survival anddifferentiation. They stimulate neurite outgrowth, suggesting that theycan promote regeneration of injured neurons, and act as target-derivedneurotrophic factors to stimulate collateral sprouting in target tissuesthat produce the neurotrophin (Korsching (1993) J. Neurosci. 13:2739).Brain-derived neurotrophic factor (BDNF) was initially characterized asa basic protein present in brain extracts and capable of increasing thesurvival of dorsal root ganglia (Leibrock et al. (1989) Nature 341:149).When axonal communication with the cell body is interrupted by injury,Schwann cells produce neurotrophic factors such as nerve growth factor(NGF) and BDNF. Neurotrophins are released from the Schwann cells anddispersed diffusely in gradient fashion around regenerating axons, whichthen extend distally along the neurotrophins' density gradient (Ide(1996) Neurosci. Res. 25:101). Local application of BDNF to transectednerves in neonatal rats has been shown to prevent massive death of motorneurons that follows axotomy (DiStefano et al. (1992) Neuron, 8:983;Oppenheim et al. (1992) Nature 360:755; and Yan et al. (1992) Nature360:753). The mRNA titer of BDNF increases to several times the normallevel four days after axotomy and reaches its maximum at 4 weeks (Meyeret al. (1992) J. Cell Biol. 119:45). Moreover, BDNF has been reported toenhance the survival of cholinergic neurons in culture (Nonomura et al.(1995) Brain Res. 683:129). In addition, nucleic acid and polypeptidessequences of BDNF orthologs in numerous species are well known in theart and include human BDNF (NM_001143805.1, NP_001137277.1,NM_001143806.1, NP_001137278.1, NM_001143807.1, NP_001137279.1,NM_001143808.1, NP_001137280.1, NM_001143809.1, NP_001137281.1,NM_001143810.1, NP_001137282.1, NM_001143811.1, NP_001137283.1,NM_001143812.1, NP_001137284.1, NM_001143813.1, NP_001137285.1,NM_001143814.1, NP_001137286.1, NM_001143815.1, NP_001137287.1,NM_001143816.1, NP_001137288.1, NM_001709.4, NP_001700.2, NM_170731.4,NP_733927.1, NM_170732.4, NP_733928.1, NM_170733.3, NP_733929.1,NM_170734.3, NP_733930.1, NM_170735.5. and NP_733931.1), chimpanzee BDNF(NM_001012441.1 and NP_001012443.1), monkey BDNF (XM_001089568.2 andXP_001089568.2), dog BDNF (NM_001002975.1 and NP_001002975.1), cow BDNF(NM_001046607.2 and NP_001040072.1), mouse BDNF (NM_001048139.1,NP_001041604.1, NM_001048141.1, NP_001041606.1, NM_001048142.1,NP_001041607.1, NM_007540.4, and NP_031566.4), rat BDNF (NM_001270630.1,NP_001257559.1, NM_001270631.1, NP_001257560.1, NM_001270632.1,NP_001257561.1, NM_001270633.1, NP_001257562.1, NM_001270634.1,NP_001257563.1, NM_001270635.1, NP_001257564.1, NM_001270636.1,NP_001257565.1, NM_001270637.1, NP_001257566.1, NM_001270638.1,NP_001257567.1, NM_012513.4, and NP_036645.2), chicken BDNF(NM_101031616.1 and NP_001026787.1), and zebrafish BDNF (NM_1131595.2and NP_571670.2). In addition, numerous anti-BDNF antibodies having avariety of characterized specificities and suitabilities for variousimmunochemical assays are commercially available and well known in theart, including antibody pa1014 from Boster Immunoleader, antibodyBDNF-#9 from DSHB Iowa, antibody 209-401-C27 from Rockland, antibodyBML-SA665 from Enzo Life Sciences, antibody EB08117 from EverestBiotech, antibody AHP1831 from AbD Serotec, antibody ANT-010 fromAlomone, and the like.

The term “binding” or “interacting” refers to an association, which maybe a stable association, between two molecules, e.g., between apolypeptide of the invention and a binding partner, due to, for example,electrostatic, hydrophobic, ionic and/or hydrogen-bond interactionsunder physiological conditions. Exemplary interactions includeprotein-protein, protein-nucleic acid, protein-small molecule, and smallmolecule-nucleic acid interactions.

The term “biological sample” when used in reference to a diagnosticassay is intended to include tissues, cells and biological fluidsisolated from a subject, as well as tissues, cells and fluids presentwithin a subject.

The term “isolated polypeptide” refers to a polypeptide, in certainembodiments prepared from recombinant DNA or RNA, or of syntheticorigin, or some combination thereof, which (1) is not associated withproteins that it is normally found within nature, (2) is isolated fromthe cell in which it normally occurs, (3) is isolated free of otherproteins from the same cellular source, (4) is expressed by a cell froma different species, or (5) does not occur in nature.

The terms “label” or “labeled” refer to incorporation or attachment,optionally covalently or non-covalently, of a detectable marker into amolecule, such as a polypeptide. Various methods of labelingpolypeptides are known in the art and may be used. Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes, fluorescent labels, heavy atoms, enzymatic labels orreporter genes, chemiluminescent groups, biotinyl groups, predeterminedpolypeptide epitopes recognized by a secondary reporter (e.g., leucinezipper pair sequences, binding sites for secondary antibodies, metalbinding domains, epitope tags). Examples and use of such labels aredescribed in more detail below. In some embodiments, labels are attachedby spacer arms of various lengths to reduce potential steric hindrance.

As used herein, the terms “neurological diseases” or “neurologicaldisorders” refers to a host of undesirable conditions affecting neuronsin the brain of a subject. Representative examples of such conditionsinclude, without limitation, Alzheimer's disease, Parkinson's disease,Huntington's disease, Pick's disease, Kuf's disease, Lewy body disease,neurofibrillary tangles, Rosenthal fibers, Mallory's hyaline, seniledementia, myasthenia gravis, Gilles de la Tourette's syndrome, multiplesclerosis (MS), amyotrophic lateral sclerosis (ALS), progressivesupranuclear palsy (PSP), epilepsy, Creutzfeldt-Jakob disease,deafness-dytonia syndrome, Leigh syndrome, Leber hereditary opticneuropathy (LHON), parkinsonism, dystonia, motor neuron disease,neuropathy-ataxia and retinitis pimentosa (NARP), maternal inheritedLeigh syndrome (MILS), Friedreich ataxia, hereditary spastic paraplegia,Mohr-Tranebjaerg syndrome, Wilson disease, sporatic Alzheimer's disease,sporadic amyotrophic lateral sclerosis, sporadic Parkinson's disease,autonomic function disorders, hypertension, sleep disorders,neuropsychiatric disorders, depression, schizophrenia, schizoaffectivedisorder, korsakoff's psychosis, mania, anxiety disorders, phobicdisorder, learning or memory disorders, amnesia or age-related memoryloss, attention deficit disorder, dysthymic disorder, major depressivedisorder, obsessive-compulsive disorder, psychoactive substance usedisorders, panic disorder, bipolar affective disorder, severe bipolaraffective (mood) disorder (BP-1), migraines, hyperactivity and movementdisorders. As used herein, the term “movement disorder” includesneurological diseases or disorders that involve the motor and movementsystems, resulting in a range of abnormalities that affect the speed,quality and ease of movement. Movement disorders are often caused by orrelated to abnormalities in brain structure and/or function. Movementdisorders include, but are not limited to (i) tremors: including, butnot limited to, the tremor associated with Parkinson's Disease,physiologic tremor, benign familial tremor, cerebellar tremor, rubraltremor, toxic tremor, metabolic tremor, and senile tremor; (ii) chorea,including, but not limited to, chorea associated with Huntington'sDisease, Wilson's Disease, ataxia telangiectasia, infection, drugingestion, or metabolic, vascular or endocrine etiology (e.g., choreagravidarum or thyrotoxicosis); (iii) ballism (defined herein as abruptlybeginning, repetitive, wide, flinging movements affecting predominantlythe proximal limb and girdle muscles); (iv) athetosis (defined herein asrelatively slow, twisting, writhing, snake-like movements and posturesinvolving the trunk, neck, face and extremities); (v) dystonia (definedherein as a movement disorder consisting of twisting, turning tonicskeletal muscle contractions, most, but not all of which are initiateddistally); (vi) paroxysmal choreoathetosis and tonic spasm; (vii) tics(defined herein as sudden, behaviorally related, irregular, stereotyped,repetitive movements of variable complexity); (viii) tardive dyskinesia;(ix) akathesia, (x) muscle rigidity, defined herein as resistance of amuscle to stretch; (xi) postural instability; (xii) bradykinesia; (xiii)difficulty in initiating movements; (xiv) muscle cramps; (xv)dyskinesias and (xvi) myoclonus.

As used herein, the term “neurodegenerative disease” or“neurodegenerative disorder” encompass a subset of neurological diseasescharacterized by involving a progressive loss of neurons or loss ofneuronal function. Accordingly, the term “neurodegeneration” refers tothe progressive loss or function of at least one neuron or neuronalcell. The ordinarily skilled artisan will appreciate that the term“progressive loss” can refer to cell death or cell apoptosis. Theordinarily skilled artisan would further appreciate that “neuronal cellloss” refers to the loss of neuronal cells. The loss of neuronal cellsmay be a result of a genetic predisposition, congenital dysfunction,apoptosis, ischemic event, immune-mediated, free-radical induced,mitochondrial dysfunction, lesion formation, misregulation or modulationof a central nervous system-specific pathway or activity, chemicalinduced, or any injury that results in a loss of neuronal cells, as wellas a progressive loss of neuronal cells. Thus, a neurodegenerativedisorder or neurodegenerative disease, as used in the current context,includes any abnormal physical or mental behavior or experience wherethe death of neuronal cells is involved in the etiology of the disorder,or is affected by the disorder. As used herein, neurodegenerativediseases encompass disorders affecting the central and peripheralnervous systems, and include such afflictions as memory loss, stroke,dementia, personality disorders, gradual, permanent or episodic loss ofmuscle control. Examples of neurodegenerative disorders or diseases forwhich the current invention can be used preferably include, but are notlimited to, Alzheimer's Disease, Parkinson's Disease, Huntington'sDisease, amyotrophic lateral sclerosis (ALS), Pick's disease, priondiseases, dystonia, dementia with Lewy bodies, multiple system atrophy,progressive supranuclear palsy, Friedreich's Ataxia, temporal lobeepilepsy, stroke, traumatic brain injury, mitochondrialencephalopathies, Guillain-Barre syndrome, multiple sclerosis, epilepsy,myasthenia gravis, chronic idiopathic demyelinating disease (CID),neuropathy, ataxia, dementia, chronic axonal neuropathy and stroke.

As used herein, the term “neuronal” or “neuron” refers to one or morecells that are a morphologic and functional unit of the brain, spinalcolumn, and peripheral nerves consisting of nerve cell bodies,dendrites, and axons. Neuronal cell types can include, but are notlimited to, typical nerve cell body showing internal structure,horizontal cell from cerebral cortex, Martinotti cell, bipolar cell,unipolar cell, Purkinje cell, and pyramidal cell of motor area ofcerebral cortex. Exemplary neuronal cells can include, but are notlimited to, cholinergic, adrenergic, noradrenergic, dopaminergic,serotonergic, glutaminergic, GABAergic, and glycinergic.

The term “treatment,” as used herein, is defined as the application oradministration of a therapeutic agent to a patient, or application oradministration of a therapeutic agent to an isolated tissue or cell linefrom a patient, who has a disease or disorder, a symptom of a disease ordisorder or a predisposition toward a disease or disorder, with thepurpose of curing, healing, alleviating, relieving, altering, remedying,ameliorating, improving or affecting the disease or disorder, thesymptoms of disease or disorder or the predisposition toward a diseaseor disorder. A therapeutic agent includes, but is not limited to,polypeptides, small molecules, peptides, peptidomimetics, nucleic acidmolecules, antibodies, ribozymes, siRNA molecules, and sense andantisense oligonucleotides described herein

As used herein, the terms “Fndc5” and “Frcp2” refer to fibronectin typeIII domain containing 5 protein and are intended to include fragments,variants (e.g., allelic variants) and derivatives thereof. Thenucleotide and amino acid sequences of mouse Fndc5, which correspond toGenbank Accession number NM_027402.3 and NP_081678.1 respectively, areset forth in SEQ ID NOs: 1 and 2. At least three splice variantsencoding distinct human Fndc5 isoforms exist (isoform 1, NM_001171941.2,NP_001165412.1; isoform 2, NM_153756.2, NP_715637.1; and isoform 3,NM_001171940.1, NP_001165411). The nucleic acid and polypeptidesequences for each isoform is provided herein as SEQ ID NOs: 3-8,respectively. Nucleic acid and polypeptide sequences of FNDC5 orthologsin organisms other than mice and human are well known and include, forexample, chimpanzee FNDC5 (XM_003949350.1, XP_003949399.1,XM_001155446.3, and XP_001155446.3), monkey FNDC5 (XM_001098747.2 andXP_001098747.2), worm FNDC5 (XM_544428.4 and XP_544428.4), rat FNDC5(XM_002729542.3 and XP_002729588.2), chicken FNDC5 (XM_417814.2;XP_417814.2), and zebrafish FNDC5 (XM_001335368.1; XP_001335404.1). Inaddition, numerous anti-BDNF antibodies having a variety ofcharacterized specificities and suitabilities for various immunochemicalassays are commercially available and well known in the art, includingantibody LS-C166197 from Lifespan Biosciences, antibodies AG-25B-0027and -0027B from Adipogen, antibody HPA051290 from Atlas Antibodies,antibodies PAN576Hu71 and Hu01 and Hu02 and Mu01 from Usen Lifesciences,antibody AP18024PU-N from Acris Antibodies, antibody OAAB05345 fromAviva Systems Biology, antibody CPBT-33932RH from Creative Biomart,antibody orb39441 from Biorbyt, antibody ab93373 from Abcam, antibodyNBP2-14024 from Novus Biologicals, antibody F4216-25 from United StatesBiological, antibody AP8746b from Abgent, and the like.

In some embodiments, fragments of Fndc5 having one or more biologicalactivities of the full-length Fndc5 protein are described and employed.Such fragments can comprise or consist of at least one fibronectindomain of an Fndc5 protein without containing the full-length Fndc5protein sequence. In some embodiments, Fndc5 fragments can comprise orconsist of a signal peptide, extracellular, fibronectin, hydrophobic,and/or C-terminal domains of an Fndc5 protein without containing thefull-length Fndc5 protein sequence. As further indicated in theExamples, Fndc5 orthologs are highly homologous and retain commonstructural domains well known in the art. In other embodiments, the term“irisin” refers to the fragment representing residues 29 or 30 to 140 ofSEQ ID NO: 2 or the corresponding residues in an FNDC5 ortholog thereof.

TABLE 1 Mouse Fndc5 cDNA Sequence SEQ ID NO: 1atg acc aca ggg ccg tga gcc tgg ccg acc cgc gcc gcg ctc cgc ctg tgg cta ggc tgcgtc tgc ttc gag ctg gtg cag gag gaa agc cac tca gcc cat gtg aac gtg acc gtc cggcac ctc aag gcc aac tct gcc gtg gtc agc tgg gat gtc ctg gag gat gaa gtg gtc attggc ttt gcc atc tct cag cag aag aag gat gtg cgg atg ctc cgg ttc att cag gag gtgaac acc acc acc cgg tcc tgc gct ctc tgg gac ctg gag gag gac aca gaa tat atc gtccat gtg cag gcc atc tcc atc cag gga cag agc cca gcc agt gag act gtg ctc ttc aagacc cca cgc gag gct gaa aag atg gcc tca aag aac aaa gat gag Gtg acc atg aag gagatg ggg agg aac cag cag ctg cga acg (ggg) gag gtg ctg atc att gtt gtg gtc ctcttc atg tgg gca ggt gtt ata gct ctc ttc tgc cgc cag tat gat atc Atc aag gac aacgag ccc aat aaa aaa aag gag aaa acc aag agc gca tca gaa acc agc Aca ccg gag catcag ggt ggg ggt ctc ctc cgc agc aag ata tgaMouse Fndc5 Amino Acid Sequence SEQ ID NO: 2M P P G P C A W P P R A A L R L W L G C V C F A L V Q A D S P S A P V NV T V R H L K A N S A V V S W D V L E D E V V I G F A I S Q Q K K D V RM L R F I Q E V N T T T R S C A L W D L E E D T E Y I V H V Q A I S I QG Q S P A S E P V L F K T P R E A E K M A S K N K D E V T M K E M G R NQ Q L R T G E V L I I V V V L F M W A G V I A L F C R Q Y D I I K D N EP N N N K E K T K S A S E T S T P E H Q G G G L L R S K IHuman Fndc5 (isoform 1) cDNA Sequence SEQ ID NO: 3    1atgctgcgct tcatccagga ggtgaacacc accacccgct catgtgccct ctgggacctg   61gaggaggata cggagtacat agtccacgtg caggccatct ccattcaggg ccagagccca  121gccagcgagc ctgtgctctt caagaccccg cgtgaggctg agaagatggc ctccaagaac  181aaagatgagg taaccatgaa agagatgggg aggaaccaac agctgcggac aggcgaggtg  241ctgatcatcg tcgtggtcct gttcatgtgg gcaggtgtca ttgccctctt ctgccgccag  301tatgacatca tcaaggacaa tgaacccaat aacaacaagg aaaaaaccaa gagtgcatca  361gaaaccagca caccagagca ccagggcggg gggcttctcc gcagcaaggt gagggcaaga  421cctgggcctg ggtgggccac cctgtgcctc atgctctggt aaHuman Fndc5 (isoform 1) Amino Acid Sequence SEQ ID NO: 4     1mlrfigevnt ttrscalwdl eedteyivhv qaisiqgqsp asepvlfktp reaekmaskn   61kdevtmkemg raqqlrtgev liivvvlfmw agvialfcrq ydiikdnepn nnkektksas  121etstpehqgg gllrskvrar pgpgwatlcl mlwHuman Fndc5 (isoform 2) cDNA Sequence SEQ ID NO: 5    1atgctgcgct tcatccagga ggtgaacacc accacccgct catgtgccct ctgggacctg   61gaggaggata cggagtacat agtccacgtg caggccatct ccattcaggg ccagagccca  121gccagcgagc ctgtgctctt caagaccccg cgtgaggctg agaagatggc ctccaagaac  181aaagatgagg taaccatgaa agagatgggg aggaacaaac agctgcggac aggcgaggtg  241ctgatcatcg tcgtggtcct gttcatgtgg gcaggtgtca ttgccctctt ctgccgccag  301tatgacatca tcaaggacaa tgaacccaat aacaacaagg aaaaaaccaa gagtgcatca  361gaaaccagca caccagagca ccagggcggg gggcttctcc gcagcaagat atgaHuman Fndc5 (isoform 2) Amino Acid Sequence SEQ ID NO: 6    1mlrfiqevnt ttrscalwdl eedteyivhv qaisiqgqsp asepvlfktp reaekmaskn   61kdevtmkemg rnqqlrtgev liivvvlfmw agvialfcrq ydiikdnepn nnkektksas  121etstpehqgg gllrski Human Fndc5 (isoform 3) cDNA Sequence SEQ ID NO: 7   1 atgctgcgct tcatccagga ggtgaacacc accacccgct catgtgccct ctgggacctg  61 gaggaggata cggagtacat agtccacgtg caggccatct ccattcaggg ccagagccca 121 gccagcgagc ctgtgctctt caagaccccg cgtgaggctg agaagatggc ctccaagaac 101 aaagatgagg taaccatgaa agagatgggg aggaaccaac agctgcggac aggcgaggtg 241 ctgatcatcg tcgtggtcct gttcatgtgg gcaggtgtca ttgccctctt ctgccgccag 301 tatgacatca ttgaagcgtg a Human Fndc5 (isoform 3) Amino Acid SequenceSEQ ID NO: 8    1mlrfiqevnt ttrscalwdl eedteyivhv qaisiqgqsp asepvlfktp reaekmaskn   61kdevtmkemg rnqqlrtgev liivvvlfmw agvialfcrq ydiieaChicken Fndc5 cDNA Sequence SEQ ID NO: 9    1atggagaaga acagggacgg acgcggcacc cctggtgtcc atctggggat ggagaaggaa   61gatgatttag agcccggtga cacgccgggg ctgcgcgaag ccctggtggc gagatgtcac  121cgctgacgcg cacccgccgg gggtatcacc gggacgggac cogtttgctc cttccggcga  181tggggagcgg tccgggccga gggctcccgg tcccgcctgg gggaaactga ggcagacggc  241ggggccgggc ggggcggggg ccgagccgcc cccgggccgg gggagggacc ggagcggggc  301tgaccagagc tgcagcgggc ggagccgggg ctaggcgggg ccgcctcccg gccgagccga  361gccgaaccga gacgcgctgc cgagggccgc cgagcccgaa gccgcccccg gccgaaccgg  421gcggcccagc cggttccggg ccccggagct ctccgcggtg ctgaacggcg ccgccgagcc  481agagggacga cggccacgga gaggctaggc ccaggcgcgg agcggagggc cgcgggggga  541tggagccctt cctgggctgc accggcgccg cgctcctgct ctgctttcag ctacgccggt  601ctgcggccgg tggaggcaga cagcccttcg gctccggtca atgtcacagt caaacacctg  661aaggcaaact cagctgtagt gacttgggac gttctggagg atgaagttgt cattggattt  721gccatttccc agcagaagaa ggacgtgcgg atgctgcgct tcatccagga ggtgaacacc  781accacccgct cctgtgccct ctgggaccta gaggaggaca ctgagtacat tgtgcatgtc  841caggccatca gcatccaagg ccagagccct gccagtgagc cagtcctctt caagaccccc  901agggaagctg agaaactggc ttctaaaaat aaagatgagg tgacaatgaa ggagatggcg  961aagaaaaacc aacagctgcg cgcaggggaa atactcatca ttgtggtggt gttgtttatg 1021tgggcagggg tgatcgccct gttctgcagg cagtacgaca tcatcaaaga caacgagccg 1081aacaacagca aggagaaagc caagagcgcc tcagagaaca gcaccaccga gcaccagggt 1141ggggggctgc tccgcagcaa gttcccaaaa aacaaaccct cagtgaacat cattgaggca 1201taa Chicken Fndc5 Amino Acid Sequence SEQ ID NO: 10    1meknrdgrgp pgvhlgmeke ddlepgdtpg lrealvarch rcrapagglt gtgpvcsfrr   61wgavraegsr srlgeteadg gagrgggraa pgpgegperg apalgraepg lggaasrpsr  121aepsraaegr rarsrprpnr aappvpgpga lrgaerrrra rgtpaperlg pgaarraagg  181wspswaapap rscsafsyag lrpveadsps apvnvtvkhl kansavvtwd vledevvigf  241aisqgkkdvr mlrfigevnt ttrscalwdl eedteyivhv qaisigggsp asepvifktp  301reaeklaskn kdevtmkema kkngglrage iliivvvlfm wagvialfcr qydlikdnep  361nnskekaksa senstpebgg ggllrsktpk nkpsvniieaZebrafish Fndc5 cDNA Sequence SEQ ID NO: 11    1atgagttctt acagtttggc agctccagtg aatgtgtcca tcagggatct gaagagcagc   61tcagccgtgg tgacatggga cacgccagac ggagagccag tcatcggctt cgccatcaca  121caacagaaga aagatgtccg catgctgcgc tttattcaag aagtgaacac caccacgcgg  181agctgtgcat tgtgggatct ggaagctgat acggattaca ttgtgcacgt tcagtctatc  241agcataagcg gggcgagtcc tgttagtgaa gctgtgcact tcaagacccc gacagaagtt  301gaaacacagg cctccaagaa caaagacgag gtgacgatgg aggaggtcgg gccgaacgct  361cagctcaggg coggagagtt catcattatt gtggtggtcc tcatcatgtg ggcaggtgtg  421atcgcactat tctgccgtca gtatgacatc attaaagaca acgaaccaaa caataacaag  481gataaagcca agaactcgtc tgaatgcagc actccagagc acacgtcagg tggcctgctg  541cgcagtaagg tataa Zebrafish Fndc5 Amino Acid Sequence SEQ ID NO: 12    1mssyslaapv nvsirdlkss savvtwdtpd gepvigfait qqkkdvrmlr fiqevntttr   61scalwdlead tdyivhvqsi sisgaspvse avhfktptev etqasknkde vtmeevgpna  121qlragefiii vvvlimwagv ialfcrqydi ikdnepnnnk dkaknssecs tpehtsggll  181rskv Fragment of Murine Fndc5 Nucleic Acid Sequence that encodesamino acid residues 29-140 of murine Fndc5 SEQ ID NO: 13  104                                               gacagcc cctcagcccc  121tgtgaacgtg accgtccggc acctcaaggc caactctgcc gtggtcagct gggatgtcct  181ggaggatgaa gtggtcattg gctttgccat ctctcagcag aagaaggatg tgcggatgct  241ccggttcatt caggaggtga acaccaccac ccggtcctgc gctctctggg acctggagga  301ggacacagaa tatatcgtcc atgtgcaggc catctccatc cagggacaga gcccagccag  361tgagcctgtg ctcttcaaga ccccacgcga ggctgaaaag atggcctcaa agaacaaaga  421tgaggtgacc atgaaggag Murine Fndc5 (residues 29-140) SEQ ID NO: 14DSPSAPVNVTVRHLKANSAVVSWDVLEDEVVIGFAISQQKKDVRMLRFIQEVNTTTRSCALWDLEEDTEYIVHVQAISIQGQSPASEPVLFKTPREAEKMASKNKDEVTMKEFrament of Human Fndc5 Nucleic Acid Sequence SEQ ID NO: 15  161                                            gacagtccct cagccccagt  181gaacgtcacc gtcaggcacc tcaaggccaa ctctgcagtg gtgagctggg atgttctgga  241ggatgaggtt gtcatcggat ttgccatctc ccagcagaag aaggatgtgc ggatgctgcg  301cttcatccag gaggtgaaca ccaccacccg ctcatgtgcc ctctgggacc tggaggagga  361tacggagtac atagtccacg tgcaggccat ctccattcag ggccagagcc cagccagcga  421gcctgtgctc ttcaagaccc cgcgtgaggc tgagaagatg gcctccaaga acaaagatga  481ggtaaccatg aaagag

It will be appreciated that specific sequence identifiers (SEQ ID NOs)have been referenced throughout the specification for purposes ofillustration and should therefore not be construed to be limiting. Anymarker of the invention, including, but not limited to, the markersdescribed in the specification and markers described herein (e.g., BDNF,Pgc1 alpha, Npas4, Err alpha, cFos, Zrc, Zif268, and the like), are wellknown in the art and can be used in the embodiments of the invention.

I. Screening Assays

Methods (also referred to herein as a “screening assay”) are providedfor identifying enhancers of the expression or activity of Fndc5 oririsin, or fragments thereof, i.e., candidate or test compounds oragents (e.g., polypeptides, peptides, peptidomimetics, small molecules(organic or inorganic) or other drugs) which promote BDNF expression.Compounds identified using assays described herein may be useful formodulating BDNF expression or activity, e.g., increasing BDNF expressionor activity. Thus, these compounds would be useful for treating orpreventing neurological diseases or disorders as BDNF is an importantneuroregulator of neuron survival.

These assays are designed to identify agents that replicate the functionof Fndc5 or irisin, fragments thereof, bind to or interact with such aprotein, or bind to or interact with other intracellular orextracellular proteins that interact with such a protein. Such compoundsmay include, but are not limited to peptides, antibodies, nucleic acidmolecules, siRNA molecules, or small organic or inorganic compounds.Such compounds may also include other cellular proteins.

Agents identified via assays such as those described herein may beuseful, for example, increasing BDNF expression or activity oractivity-induced gene expression in the central and/or peripheralnervous system and, for example, increasing neuronal survival,decreasing lesion formation, increasing neurite growth and/or synapses,decreasing mitochondrial dysfunction, increasing neuronaldifferentiation, modulating neuronal migration, increasing dendriticarborization, increasing synaptic plasticity. Thus, these compoundswould be useful for treating or preventing a neurological disease ordisorder, particularly neurodegenerative diseases or disorders. In someembodiments, increased activity or expression of Fndc5 or irisin,fragments thereof, would bring about an effective increase in the levelof BDNF protein activity, thus identifying, treating or preventingneurological diseases or disorders. For example, a partial agonist or anagonist administered in a dosage or for a length of time to increaseexpression or activity of Fndc5 or irisin, or fragments thereof wouldact to increase neuronal survival, decrease lesion formation, increaseneurite outgrowth and/or synapses, increase mitochondrial function, andtreat or prevent a neurological disease or disorder.

In one embodiment, the invention provides assays for screening candidateor test compounds which are substrates of or interact with Fndc5 oririsin, or fragments thereof. In another embodiment, the inventionprovides assays for screening candidate or test compounds which bind toor modulate the activity of Fndc5 or irisin, or fragments thereof. Instill another embodiment, the invention provides assays for screeningcandidate Fndc5 or irisin proteins, or fragments thereof, having desiredfunctional characteristics. The test agents of the present invention canbe obtained using any of the numerous approaches in combinatoriallibrary methods known in the art, including: biological libraries,spatially addressable parallel solid phase or solution phase libraries;synthetic library methods requiring deconvolution; the ‘one-beadone-compound’ library method; and synthetic library methods usingaffinity chromatography selection. The biological library approach islimited to polypeptide or peptide libraries, while the other fourapproaches are applicable to peptide, non-peptide oligomer or smallmolecule libraries of compounds (Lam, K. S. (1997) Anticancer Drug Des.12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. USA 91:11422;Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al. (1993)Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed. Engl.33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; andin Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids(Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage(Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci.87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladnersupra.).

In one embodiment, an assay is a cell-based assay in which a cell, suchas a neuron, is contacted with a test agent, such as an Fndc5 or irisinpolypeptide, or fragments thereof, and the ability of the test compoundto modulate BDNF expression or activity is determined. Determining theability of the test agent to modulate BDNF expression or activity can beaccomplished by monitoring, for example, neuronal survival, BDNFexpression levels, the level of transcription of genes downstream ofBDNF, and the like. The cell can be of mammalian origin, e.g., a neuron.

The ability of the test agent to modulate the binding of Fndc5 or irisinpolypeptide, or fragments thereof, to a substrate such as a modulator ofBDNF expression (e.g., NPas4 or other upstream gene or protein) can alsobe determined. Determining the ability of the test agent to modulatesuch binding can be accomplished, for example, by coupling the substratewith a radioisotope or enzymatic label such that binding of thesubstrate to Fndc5 or irisin polypeptide, or fragments thereof can bedetermined by detecting the labeled substrate in a complex. The Fndc5 oririsin polypeptide, fragments thereof, can also be coupled with aradioisotope or enzymatic label to monitor the ability of a test agentto modulate binding to the substrate in a complex. Determining theability of the test agent to bind Fndc5 or irisin polypeptide, orfragments thereof, can be accomplished, for example, by coupling theagent with a radioisotope or enzymatic label such that binding of theagent to Fndc5 or irisin polypeptide, or fragments thereof, can bedetermined by detecting the labeled agent in a complex. For example,such agents can be labeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directlyor indirectly, and the radioisotope detected by direct counting ofradioemmission or by scintillation counting. Agents can further beenzymatically labeled with, for example, horseradish peroxidase,alkaline phosphatase, luciferase, and the enzymatic label detected bydetermination of conversion of an appropriate substrate to product.

It is also within the scope of the present invention to determine theability of an agent to interact with Fndc5 or irisin polypeptide, orfragments thereof, or with a modulator BDNF expression or activity,without the labeling of any of the interactants. For example, amicrophysiometer can be used to detect the interaction without labelingany component (McConnell, H. M. et al. (1992) Science 257:1906-1912. Asused herein, a “microphysiometer” (e.g., Cytosensor) is an analyticalinstrument that measures the rate at which a cell acidifies itsenvironment using a light-addressable potentiometric sensor (LAPS).

In another embodiment, modulators of BDNF expression are identified in amethod wherein a cell is contacted with a candidate agent, such as Fndc5or irisin polypeptide, or fragments thereof, and the expression of BDNFmRNA or protein in the cell is determined. The level of expression ofBDNF mRNA or protein in the presence of the candidate agent is comparedto the level of expression of BDNF mRNA or protein in the absence of thecandidate agent. When expression of BDNF mRNA or protein is greater(statistically significantly greater) in the presence of the candidateagent than in its absence, the candidate agent is identified as astimulator of BDNF mRNA or protein expression. The level of BDNF mRNA orprotein expression in the cells can be determined by methods describedherein for detecting BDNF mRNA or protein.

In some embodiments, the assays can be conducted in cell-free formatsusing known components of BDNF gene expression (e.g., Npas4). It may bedesirable to immobilize certain components of the assay, such as theFndc5 or irisin polypeptide, or fragments thereof and such embodimentsmay benefit from the use of well-known adaptations for biomoleculeimmobilization, such as the use of microtiter plates, beads, test tubes,micro-centrifuge tubes in combination with derivatizable moieties, suchas fusion protein domains, biotinylzation, antibodies, and the like.

The present invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model.

Any of the compounds, including but not limited to compounds such asthose identified in the foregoing assay systems, may be tested for acompound capable of treating or preventing a neurological disease ordisorder comprising the ability of the compound to modulate BDNF nucleicacid expression or BDNF polypeptide activity, thereby identifying acompound capable of treating or preventing a neurological disease ordisorder. Cell-based and animal model-based assays for theidentification of compounds exhibiting such an ability to treat orprevent a neurological disease or disorder described herein.

In one aspect, cell-based systems, described herein, may be used toidentify agents such as an Fndc5 or irisin polypeptide, or fragmentsthereof, that modulate BDNF nucleic acid expression or BDNF polypeptideactivity or treat neurological diseases or disorders. For example, suchcell systems may be exposed to an agent at a sufficient concentrationand for a time sufficient to elicit such an amelioration of diseasesymptoms in the exposed cells. After exposure, the cells are examined todetermine whether one or more of the disease phenotypes, e.g., neuronalsurvival, for example, has been altered to resemble a more normal ormore wild type disease phenotype.

In addition, animals or animal-based disease systems, such as thosedescribed herein, may be used to identify such agents. Such animalmodels may be used as test substrates for the identification of drugs,pharmaceuticals, therapies, and interventions which may be effective inmodulating PGC-1α, treating or preventing neurological diseases ordisorders. In some embodiments, the parameters of the assay are definedto allow for systemic or serum expression of the agent to cross theblood-brain barrier.

Additionally, gene expression patterns may be utilized to assess theability of a compound to modulate BDNF expression or activity. Thus,these compounds would be useful for treating, preventing, or assessing aneurological disease or disorder. For example, the expression pattern ofone or more genes may form part of a “gene expression profile” or“transcriptional profile” which may be then be used in such anassessment. “Gene expression profile” or “transcriptional profile”, asused herein, includes the pattern of mRNA expression obtained for agiven tissue or cell type wider a given set of conditions. Geneexpression profiles may be generated, for example, by utilizing adifferential display procedure. Northern analysis and/or RT-PCR. Geneexpression profiles may be characterized for known states within thecell- and/or animal-based model systems. Subsequently, these known geneexpression profiles may be compared to ascertain the effect a testcompound has to modify such gene expression profiles, and to cause theprofile to more closely resemble that of a more desirable profile. Forexample, useful markers are described herein and include, withoutlimitation, markers of mitochondrial function such as LDH2, Ndufb5,COX6a1, and ATP5j, markers of neuronal activity, such as immediate earlygenes, NF-H, NF-M, MOBP, ATPa1, and ATP1a2, upstream and downstreamregulators of BDNF gene expression, and the like.

II. Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating or preventing a neurological disease or disorder ina subject, e.g., a human, at risk of (or susceptible to) a neurologicaldisease or disorder, by administering to said subject an enhancer ofBDNF expression or activity, such as Fndc5 or irisin polypeptide, orfragments thereof, such that the neurological disease or disorder istreated or prevented. In some embodiments, which includes bothprophylactic and therapeutic methods, the BDNF modulator is administeredby in a pharmaceutically acceptable formulation.

With regard to both prophylactic and therapeutic methods of treatment,such treatments may be specifically tailored or modified, based onknowledge obtained from the field of pharmacogenomics.“Pharmacogenomics,” as used herein, refers to the application ofgenomics technologies such as gene sequencing, statistical genetics, andgene expression analysis to drugs in clinical development and on themarket. More specifically, the term refers to the study of how apatient's genes determine his or her response to a drug (e.g., apatient's “drug response phenotype”, or “drug response genotype”).

Thus, another aspect of the invention provides methods for tailoring asubject's prophylactic or therapeutic treatment with either Fndc5 oririsin polypeptide, or fragments thereof, or other BDNF modulatorsaccording to that individual's drug response genotype. Pharmacogenomicsallows a clinician or physician to target prophylactic or therapeutictreatments to patients who will most benefit from the treatment and toavoid treatment of patients who will experience toxic drug-related sideeffects.

A. Prophylactic Methods

In one aspect, the present invention provides a method for treating orpreventing a neurological disease or disorder by administering to asubject an agent which modulates BDNF expression or activity in thecentral and/or peripheral nervous system using an Fndc5 or irisinpolypeptide, or fragments thereof, or an enhancer of such apolypeptide's expression or activity. The present invention alsoprovides methods for modulating neuronal survival, formation of brainlesions, neurodegeneration, and/or neurite/synapse growth in thesubject. Subjects at risk for a neurological disease or disorder can beidentified by, for example, any or a combination of the diagnostic orprognostic assays described herein. Administration of a prophylacticagent can occur prior to the manifestation of symptoms characteristic ofa neurological disease or disorder, such that the neurological diseaseor disorder or symptom thereof, e.g., neuronal cell death, is preventedor, alternatively, delayed in its progression.

B. Therapeutic Methods

The present invention provides methods for modulating BDNF expression oractivity in the central and/or peripheral nervous system in a subject byadministering an agent which modulates BDNF expression or activity inthe central and/or peripheral nervous system using an Fndc5 or irisinpolypeptide/nucleic acid, or fragments thereof, or an enhancer of such apolypeptide/nucleic acid expression or activity. In one embodiment, BDNFexpression or activity is increased by administering an inducer oragonist of BDNF expression or activity, thereby modulating modulatingneuronal survival, formation of brain lesions, neurodegeneration, and/orneurite/synapse growth in the subject.

Accordingly, another aspect of the invention pertains to methods ofmodulating BDNF expression or activity for therapeutic purposes and foruse in treatment of neurological diseases or disorders. In an exemplaryembodiment, the modulatory method of the invention involves contacting acell with an Fndc5 or irisin polypeptide, or fragments thereof, or anenhancer of such a polypeptide's or nucleic acid's expression oractivity. In one embodiment, the agent simulates one or more activitiesof an Fndc5 or irisin polypeptide, or fragments thereof, or an enhancerof such a polypeptide's expression or activity. Examples of suchsimulator agents include small molecule agonists and mimetics, e.g., apeptidomimetic. These modulatory methods can be performed in vitro or exvivo (e.g., by culturing the cell with the agent) or, alternatively, invivo (e.g., by administering the agent to a subject). In one embodiment,the method involves administering an agent (e.g., an agent identified bya screening assay described herein), or combination of agents thatmodulate BDNF expression or activity or are otherwise useful fortreating or preventing neurological diseases or disorders, such asneurotrophic factors, free radical inhibitors, and the like.

Increasing BDNF expression or activity leads to treatment or preventionof a neurological disease or disorder, therefore providing a method fortreating, preventing, and assessing a neurological disease or disorder.A variety of techniques may be used to increase the expression,synthesis, or activity of BDNF using an Fndc5 or irisin polypeptide, orfragments thereof, or an enhancer of such a polypeptide's expression oractivity.

For example, an Fndc5 or irisin polypeptide/nucleic acid, or fragmentsthereof, or an enhancer of such a polypeptide/nucleic acid expression oractivity protein may be administered to a subject. Any of the techniquesdiscussed below may be used for such administration. One of skill in theart will readily know how to determine the concentration of effective,non--toxic doses of the protein, utilizing techniques such as thosedescribed below.

Additionally, nucleic acid sequences, such as RNA sequences encodingsuch proteins may be directly administered to a subject, at aconcentration sufficient to produce a level of an Fndc5or irisinpolypeptide, or fragments thereof, or an enhancer of such apolypeptide's expression or activity, such that BDNF expression oractivity in the peripheral and/or central nervous system is modulated.Any of the techniques discussed below, which achieve intracellularadministration of compounds, such as, for example, liposomeadministration, may be used for the administration of such nucleic acidmolecules. RNA molecules may be produced, for example, by recombinanttechniques such as those described herein. Other pharmaceuticalcompositions, medications, or therapeutics may be used in combinationwith the agents described herein. Further, subjects may be treated bygene replacement therapy. For example, one or more copies of an Fndc5 oririsin polypeptide, or fragments thereof, or an enhancer of such apolypeptide's expression or activity, may be inserted into cells usingvectors which include, but are not limited to adenovirus,adeno-associated virus, and retrovirus vectors, in addition to otherparticles that introduce DNA into cells, such as liposomes.Additionally, techniques such as those described above may be used forthe introduction of desired gene sequences into human cells.Furthermore, expression or activity of transcriptional activators whichact upon BDNF may be increased to thereby increase expression andactivity of BDNF. Small molecules enhance the expression or activity ofan Fndc5 or irisin polypeptide, or fragments thereof, either directly orindirectly may also be used.

Cells, preferably, autologous cells, containing PGC-1α expressing genesequences may then be introduced or reintroduced into the subject. Suchcell replacement techniques may be preferred, for example, when the geneproduct is a secreted, extracellular gene product.

C. Pharmaceutical Compositions

The methods of the invention involve administering to a subject an agentwhich modulates BDNF expression or activity in the central and/orperipheral nervous system in a subject by administering an agent whichmodulates BDNF expression or activity in the central and/or peripheralnervous system using an Fndc5 or irisin polypeptide, or fragmentsthereof, or an enhancer of such a polypeptide's expression or activity,either alone or in combination with other agents useful for treating orpreventing an undesirable neurological disorder or condition.

The agents which modulate BDNF expression or activity can beadministered in a therapeutically effective amount to a subject usingpharmaceutical compositions suitable for such administration. Suchcompositions typically comprise the agent (e.g., nucleic acid moleculeor protein) and a pharmaceutically acceptable carrier. As used hereinthe language “pharmaceutically acceptable carrier” is intended toinclude any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the compositionsis contemplated. Supplementary active compounds can also be incorporatedinto the compositions.

The term “effective amount” of an agent that induces expression and/oractivity of Fndc5 is that amount necessary or sufficient to modulate(e.g., increase or decrease) expression and/or activity of Fndc5 in thesubject or population of subjects. The effective amount can varydepending on such factors as the type of therapeutic agent(s) employed,the size of the subject, or the severity of the disorder.

The term “therapeutically effective amount” as used herein means thatamount of an agent that modulates (e.g., enhances) the expression oractivity of Fndc5 or irisin, or fragments thereof, or compositioncomprising an agent that modulates (e.g., enhances) such expression oractivity, which is effective for producing some desired therapeuticeffect, e.g., BDNF expression in the central and/or peripheral nervoussystem, at a reasonable benefit/risk ratio.

A pharmaceutical composition used in the therapeutic methods of theinvention is formulated to be compatible with its intended route ofadministration. Examples of routes of administration include parenteral,e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation),transdermal (topical), transmucosal, and rectal administration.Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylenediaminetetraacetic acid; buffers suchas acetates, citrates or phosphates and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringesor multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, and sodium chloride inthe composition. Prolonged absorption of the injectable compositions canbe brought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the agentthat modulates BDNF activity (e.g., Fndc5 or irisin polypeptide, orfragments thereof, or an enhancer of such a polypeptide's expression oractivity) in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The agents that modulate BDNF expression or activity can also beprepared in the form of suppositories (e.g., with conventionalsuppository bases such as cocoa butter and other glycerides) orretention enemas for rectal delivery.

In one embodiment, the agents that modulate BDNF expression or activityare prepared with carriers that will protect the compound against rapidelimination from the body, such as a controlled release formulation,including implants and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Methods for preparation of suchformulations will be apparent to those skilled in the art. The materialscan also be obtained commercially from Alza Corporation and NovaPharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to infected cells with monoclonal antibodies to viral antigens)can also be used as pharmaceutically acceptable carriers. These can beprepared according to methods known to those skilled in the art, forexample, as described in U.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for case of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the agent that modulatesPGC-1α activity and the particular therapeutic effect to be achieved,and the limitations inherent in the art of compounding such an agent forthe treatment of subjects.

Toxicity and therapeutic efficacy of such agents can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and can be expressed as the ratio L50/ED50. Agentswhich exhibit large therapeutic indices are preferred. While agents thatexhibit toxic side effects may be used, care should be taken to design adelivery system that targets such agents to the site of affected tissuein order to minimize potential damage to uninfected cells and, thereby,reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch BDNF modulating agents lies preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anyagent used in the therapeutic methods of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range that includes the IC50 (i.e., theconcentration of the test compound which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

As defined herein, a therapeutically effective amount of protein orpolypeptide (i.e., an effective dosage) ranges from about 0.001 to 30mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, morepreferably about 0.1 to 20 mg/kg body weight, and even more preferablyabout 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6mg/kg body weight. The skilled artisan will appreciate that certainfactors may influence the dosage required to effectively treat asubject, including but not limited to the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject, and other diseases present. Moreover, treatment of a subjectwith a therapeutically effective amount of a protein, polypeptide, orantibody can include a single treatment or, preferably, can include aseries of treatments.

In a preferred example, a subject is treated with a polypeptide in therange of between about 0.1 to 20 mg/kg body weight, one time per weekfor between about 1 to 10 weeks, preferably between 2 to 8 weeks, morepreferably between about 3 to 7 weeks, and even more preferably forabout 4, 5, or 6 weeks. It will also be appreciated that the effectivedosage of antibody, protein, or polypeptide used for treatment mayincrease or decrease over the course of a particular treatment. Changesin dosage may result and become apparent from the results of diagnosticassays as described herein.

The present invention encompasses agents which modulate expression oractivity. An agent may, for example, be a small molecule. For example,such small molecules include, but are not limited to, peptides,peptidomimetics, amino acids, amino acid analogs, polynucleotides,polynucleotide analogs, nucleotides, nucleotide analogs, organic orinorganic compounds (i.e., including heteroorganic and organometalliccompounds) having a molecular weight less than about 10,000 grains permole, organic or inorganic compounds having a molecular weight less thanabout 5,000 grams per mole, organic or inorganic compounds having amolecular weight less than about 1,000 grams per mole, organic orinorganic compounds baying a molecular weight less than about 500 gramsper mole, and salts, esters, and other pharmaceutically acceptable formsof such compounds. It is understood that appropriate doses of smallmolecule agents depends upon a number of factors within the ken of theordinarily skilled physician, veterinarian, or researcher. The dose(s)of the small molecule will vary for example, depending upon theidentity, size, and condition of the subject or sample being treated,further depending upon the route by which the composition is to beadministered, if applicable, and the effect which the practitionerdesires the small molecule to have upon the nucleic acid or polypeptideof the invention.

Exemplary doses include milligram or microgram amounts of the smallmolecule per kilogram of subject or sample weight (e.g., about 1microgram per kilogram to about 500 milligrams per kilogram, about 100micrograms per kilogram to about 5 milligrams per kilogram, or about 1microgram per kilogram to about 50 micrograms per kilogram). It isfurthermore understood that appropriate doses of a small molecule dependupon the potency of the small molecule with respect to the expression oractivity to be modulated. Such appropriate doses may be determined usingthe assays described herein. When one or more of these small moleculesis to be administered to an animal. (e.g., a human) in order to modulateexpression or activity of a BDNF molecule, a physician, veterinarian, orresearcher may, for example, prescribe a relatively low dose at first,subsequently increasing the dose until an appropriate response isobtained. In addition, it is understood that the specific dose level forany particular animal subject will depend upon a variety of factorsincluding the activity of the specific compound employed, the age, bodyweight, general health, gender, and diet of the subject, the time ofadministration, the route of administration, the rate of excretion, anydrug combination, and the degree of expression or activity to bemodulated, e.g., the intended use of the agonist or antagonize.

Further, the BDNF modulating agents described herein can be conjugatedto additional therapeutic moieties of interest, such as a growth factor,intracellular targeting domain, and the like, that are well known in theart. The conjugates of the invention can be used for modifying a givenbiological response, the drug moiety is not to be consulted as limitedto classical chemical therapeutic agents. For example, the drug moietymay be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, a toxin such as abrin,ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such astumor necrosis factor, alpha-interferon, beta-interferon, nerve growthfactor, platelet derived growth factor, tissue plasminogen activator; orbiological response modifiers such as, for example, lymphokines,interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”),granulocyte macrophase colony stimulating factor (“GM-CSF”), granulocytecolony stimulating factor (“G-CSF”), or other growth factors.

The nucleic acid molecules used in the methods of the invention can beinserted into vectors and used as gene therapy vectors. Gene therapyvectors can be delivered to a subject by for example, intravenousinjection, local administration (see U.S. Pat. No. 5,328,470) or bystereotactic injection (see, e.g., Chen et al. (1994) Proc. Natl. Acad.Sci. USA 91:3054-3057). The pharmaceutical preparation of the genetherapy vector can include the gene therapy vector in an acceptablediluent, or can comprise a slow release matrix in which the genedelivery vehicle is imbedded. Alternatively, where the complete genedelivery vector can be produced intact from recombinant cells, e.g.,retroviral vectors, the pharmaceutical preparation can include one ormore cells which produce the gene delivery system.

Any means for the introduction of a polynucleotide into mammals, humanor non-human or cells thereof may be adapted to the practice of thisinvention for the delivery of the various constructs of the inventioninto the intended recipient. In one embodiment of the invention, the DNAconstructs are delivered to cells by transfection, i.e., by delivery of“naked” DNA or in a complex with a colloidal dispersion system. Acolloidal system includes macromolecule complexes, nanocapsules,microspheres, beads, and lipid-based systems including oil-in-wateremulsions, micelles, mixed micelles, and liposomes. The preferredcolloidal system of this invention is a lipid-complexed orliposome-formulated DNA. In the former approach, prior to formulation ofDNA, e.g., with lipid, a plasmid containing a transgene bearing thedesired DNA constructs may first be experimentally optimized forexpression (e.g., inclusion of an intron in the 5′ untranslated regionand elimination of unnecessary sequences (Feigner, et al., Ann NY AcadSci 126-139, 1995). Formulation of DNA, e.g. with various lipid orliposome materials, may then be effected using known methods andmaterials and delivered to the recipient mammal. See, e.g., Canonico etal, Am Respir Cell Mol Biol 10:24-29, 1994: Tsan et al. Am J Physiol268: Alton et al., Nat Genet. 5:135-142, 1993 and U.S. Pat. No.5,679,647 by Carson et al.

The targeting of liposomes can be classified based on anatomical andmechanistic factors. Anatomical classification is based on the level ofselectivity, for example, organ-specific, cell-specific, andorganelle-specific. Mechanistic targeting can be distinguished basedupon whether it is passive or active. Passive targeting utilizes thenatural tendency of liposomes to distribute to cells of thereticulo-endothelial system (RES) in organs, which contain sinusoidalcapillaries. Active targeting, on the other hand, involves alteration ofthe liposome by coupling the liposome to a specific ligand such as amonoclonal antibody, sugar, glycolipid, or protein, or by changing thecomposition or size of the liposome in order to achieve targeting toorgans and cell types other than the naturally occurring sites oflocalization.

The surface of the targeted delivery system may be modified in a varietyof ways. In the case of a liposomal targeted delivery system, lipidgroups can be incorporated into the lipid bilayer of the liposome inorder to maintain the targeting ligand in stable association with theliposomal bilayer. Various linking groups can be used for joining thelipid chains to the targeting ligand. Naked DNA or DNA associated with adelivery vehicle, e.g., liposomes, can be administered to several sitesin a subject (see below).

Nucleic acids can be delivered in any desired vector. These includeviral or non-viral vectors, including adenovirus vectors,adeno-associated virus vectors, retrovirus vectors, lentivirus vector,and plasmid vectors. Exemplary types of viruses include HSV (herpessimplex virus), AAV (adeno associated virus), HIV (humanimmunodeficiency virus), BIV (bovine immunodeficiency virus), and MLV(murine leukemia virus). Nucleic acids can be administered in anydesired format that provides sufficiently efficient delivery levels,including in virus particles, in liposomes, in nanoparticles, andcomplexed to polymers.

The nucleic acids encoding a protein or nucleic acid of interest may bein a plasmid or viral vector, or other vector as is known in the art.Such vectors are well known and any can be selected for a particularapplication. In one embodiment of the invention, the gene deliveryvehicle comprises a promoter and a demethylase coding sequence.Preferred promoters are tissue-specific promoters and promoters whichare activated by cellular proliferation, such as the thymidine kinaseand thymidylate synthase promoters. Other preferred promoters includepromoters which are activatable by infection with a virus, such as theα- and β-interferon promoters, and promoters which are activatable by ahormone, such as estrogen. Other promoters which can be used include theMoloney virus LTR, the CMV promoter, and the mouse albumin promoter. Apromoter may be constitutive or inducible.

In another embodiment, naked polynucleotide molecules are used as genedelivery vehicles, as described in WO 9011092 and U.S. Pat. No.5,580,859. Such gene delivery vehicles can be either growth factor DNAor RNA and, in certain embodiments, are linked to killed adenovirus.Curiel et al., Hum. Gene. Ther. 3:147-154, 1992. Other vehicles whichcan optionally be used include DNA-ligand (Wu et al., J. Biol. Chem.264:16985-16987, 1989), lipid-DNA combinations (Felgner et al., Proc.Natl. Acad. Sci. USA 84:7413 7417, 1989), liposomes (Wang et al., Proc.Natl. Acad. Sci. 84:7851-7855, 1987) and microprojectiles (Williams etal., Proc. Natl. Acad. Sci. 88:2726-2730, 1991).

A gene delivery vehicle can optionally comprise viral sequences such asa viral origin of replication or packaging signal. These viral sequencescan be selected from viruses such as astrovirus, coronavirus,orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus,poxvirus, retrovirus, togavirus or adenovirus. In a preferredembodiment, the growth factor gene delivery vehicle is a recombinantretroviral vector. Recombinant retroviruses and various uses thereofhave been described in numerous references including, for example, Mannet al., Cell 33:153, 1983, Cane and Mulligan, Proc. Nat'l. Acad. Sci.USA 81:6349, 1984, Miller et al., Human Gene Therapy 1:5-14, 1990, U.S.Pat. Nos. 4,405,712, 4,861,719, and 4,980,289, and PCT Application Nos.WO 89/02,468, WO 89/05,349, and WO 90/02,806. Numerous retroviral genedelivery vehicles can be utilized in the present invention, includingfor example those described in EP 0,415,731; WO 90/07936; WO 94/03622;WO 93/25698; WO 93/25234; U.S. Pat. No. 5,219,740; WO 9311230; WO9310218; Vile and Hart, Cancer Res. 53:3860-3864, 1993; Vile and Hart,Cancer Res. 53:962-967, 1993; Ram et al., Cancer Res. 53:83-88, 1993;Takamiya et al., J. Neurosci. Res. 33:493-503, 1992; Baba et al., J.Neurosurg. 79:729-735, 1993 (U.S. Pat. No. 4,777,127, GB 2,200,651, EP0,345,242 and WO91/02805).

Other viral vector systems that can be used to deliver a polynucleotideof the invention have been derived from herpes virus, e.g., HerpesSimplex Virus (U.S. Pat. No. 5,631,236 by Woo et al., issued May 20,1997 and WO 00/08191 by Neurovex), vaccinia virus (Ridgeway (1988)Ridgeway, “Mammalian expression vectors,” In: Rodriguez R L, Denhardt DT, ed. Vectors: A survey of molecular cloning vectors and their uses.Stoneham: Butterworth: Baichwal and Sugden (1986) “Vectors for genetransfer derived from animal DNA viruses: Transient and stableexpression of transferred genes,” In: Kucherlapati R, ed. Gene transfer.New York: Plenum Press; Coupar et al. (1988) Gene, 68:1-10), and severalRNA viruses. Preferred viruses include an alphavirus, a poxivirus, anarena virus, a vaccinia virus, a polio virus, and the like. They offerseveral attractive features for various mammalian cells (Friedmann(1989) Science, 244:1275-1281; Ridgeway, 1988, supra; Baichwal andSugden, 1986, supra; Coupar et al., 1988; Horwich et al. (1990) J.Virol., 64:642-650).

In other embodiments, target DNA in the genome can be manipulated usingwell-known methods in the art. For example, the target DNA in the genomecan be manipulated by deletion, insertion, and/or mutation areretroviral insertion, artificial chromosome techniques, gene insertion,random insertion with tissue specific promoters, gene targeting,transposable elements and/or any other method for introducing foreignDNA or producing modified DNA/modified nuclear DNA. Other modificationtechniques include deleting DNA sequences from a genome and/or alteringnuclear DNA sequences. Nuclear DNA sequences, for example, may bealtered by site-directed mutagenesis.

In other embodiments, recombinant Fndc5 polypeptides, and fragmentsthereof, can be administered to subjects. In some embodiments, fusionproteins can be constructed and administered which have enhancedbiological properties (e.g., Fc fusion proteins discussed above). Inaddition, the Fndc5 polypeptides, and fragment thereof, can be modifiedaccording to well known pharmacological methods in the art (e.g.,pegylation, glycosylation, oligomerization, etc.) in order to furtherenhance desirable biological activities, such as increasedbioavailability and decreased proteolytic degradation.

III. Predictive Medicine

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, and monitoring ofclinical trials are used for prognostic (predictive) purposes to therebytreat an individual prophylactically. Accordingly, one aspect of thepresent invention relates to diagnostic assays for determining thelevels of protein and/or nucleic acid expression or activity of a BDNFand/or Fndc5 or irisin polypeptide, or fragments thereof, in the contextof a biological sample (e.g., blood, serum, fluid, e.g., cerebrospinalfluid, spinal fluid, cells, or tissue, e.g., neural tissue) to therebydetermine whether an individual is afflicted with a neurological diseaseor disorder neurological disease or disorder has a risk of developing aneurological disease or disorder. The invention also provides forprognostic (or predictive) assays for determining whether an individualis at risk of developing a neurological disease or disorder.

One particular embodiment includes a method for assessing whether asubject is afflicted with a neurological disease or disorder or is atrisk of developing a neurological disease or disorder comprisingdetecting the expression or activity of the Fndc5 or irisin polypeptide,or fragments thereof in a cell or tissue sample of a subject, wherein adecrease in the expression or activity thereof indicates the presence ofa neurological disease or disorder or the risk of developing aneurological disease or disorder in the subject. In this embodiment,subject samples tested are, for example, cerebrospinal fluid, spinalfluid, and neural tissue.

Another aspect of the invention pertains to monitoring the influence ofmodulators of BDNF expression in clinical trials.

These and other agents are described in further detail in the followingsections.

A. Prognostic and Diagnostic Assays

To determine whether a subject is afflicted with a neurological diseaseor disorder has a risk of developing, a neurological disease ordisorder, a biological sample may be obtained from a subject and thebiological sample may be contacted with a compound or an agent capableof detecting an Fndc5 or irisin polypeptide, or fragments thereof, ornucleic acid (e.g., mRNA or genomic DNA) that encodes such a protein, inthe biological sample. A preferred agent for detecting the mRNA orgenomic DNA is a labeled nucleic acid probe capable of hybridizing tothe mRNA or genomic DNA. The nucleic acid probe can be, for example, asequence that is complementary to an Fndc5 or irisin nucleic acid setforth in Table 1, or a portion thereof, such as an oligonucleotide of atleast 15, 20, 25, 30, 25, 40, 45, 50, 100, 250 or 500 nucleotides inlength and sufficient to specifically hybridize under stringentconditions to the desired mRNA or genomic DNA. Other suitable probes foruse in the diagnostic assays of the invention are described herein.

The term “biological sample” is intended to include tissues, cells, andbiological fluids isolated from a subject, as well as tissues, cells,and fluids present within a subject, e.g., cerebrospinal fluid, spinalfluid, and neural tissue. That is, the detection method of the inventioncan be used to detect mRNA, protein, or genomic DNA of Fndc5 or irisin,or portions thereof, in a biological sample in vitro as well as in vivo.For example, in vitro techniques for detection of mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetection of protein include enzyme linked immunosorbent assays(ELISAs), Western blots, immunoprecipitations and immunofluorescence. Invitro techniques for detection of genomic DNA include Southernhybridizations. Furthermore, in vivo techniques for detection of proteininclude introducing into a subject a labeled antibody against thedesired protein to be detected. For example, the antibody can be labeledwith a radioactive marker whose presence and location in a subject canbe detected by standard imaging techniques.

In another embodiment, the methods further involve obtaining a controlbiological sample from a control subject, contacting the control samplewith a compound or agent capable of detecting protein, mRNA, or genomicDNA, such that the presence of the desired protein, mRNA or genomic DNAis detected in the biological sample, and comparing the presence of theprotein, mRNA or genomic DNA in the control sample with the presence ofthe protein, mRNA or genomic DNA in the rest sample.

Analysis of one or more polymorphic regions of Fndc5 or irisin nucleicacids, or fragments thereof in a subject can be useful for predictingwhether a subject has or is likely to develop a neurological disease ordisorder. In preferred embodiments, the methods of the invention can becharacterized as comprising detecting, in a sample of cells from thesubject, the presence or absence of a specific allelic variant of one ormore polymorphic regions of the gene, such as a premature truncationthat does not encode a biologically active protein or a mutation in thestop codon or other region that prevents protein access across theblood-brain barrier. The allelic differences can be: (i) a difference inthe identity of at least one nucleotide or (ii) a difference in thenumber of nucleotides, which difference can be a single nucleotide orseveral nucleotides. The invention also provides methods for detectingdifferences in an Fndc5- or irisin-encoding gene such as chromosomalrearrangements, e.g., chromosomal dislocation. The invention can also beused in prenatal diagnostics.

A preferred detection method is allele specific hybridization usingprobes overlapping the polymorphic site and having about 5, 10, 20, 25,or 30 nucleotides around the polymorphic region. In a preferredembodiment of the invention, several probes capable of hybridizingspecifically to allelic variants are attached to a solid phase supporte.g., a “chip”. Oligonucleotides can be bound to a solid support by avariety of processes, including lithography. For example, a chip canhold up to 250,000 oligonucleotides (GeneChip, Affymetrix). Mutationdetection analysis using, these chips comprising oligonucleotides, alsotermed “DNA probe arrays” is described e.g., in Cronin et al. (1996)Human Mutation 7:244. In one embodiment, a chip comprises all theallelic variants of at least one polymorphic region of a gene. The solidphase support is then contacted with a test nucleic acid andhybridization to the specific probes is detected. Accordingly, theidentity of numerous allelic variants of one or more genes can beidentified in a simple hybridization experiment. For example, theidentity of the allelic variant of the nucleotide polymorphism in the 5′upstream regulatory element can be determined in a single hybridizationexperiment.

In other detection methods, it is necessary to first amplify at least aportion of nucleic acid prior to identifying the allelic variant.Amplification can be performed, e.g., by PCR and/or LCR (see Wu andWallace, (1989) Genomics 4:560), according to methods known in the art.In one embodiment, genomic DNA of a cell is exposed to two PCR primersand amplification for a number of cycles sufficient to produce therequired amount of amplified DNA. In preferred embodiments, the primersare located between 150 and 350 base pairs apart.

Alternative amplification methods include: self sustained sequencereplication (Guatelli, J. C. et al., 1990, Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh, D. Y. 1989,Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi, P.M. et al., 1988, Bio/Technology 6:1197), and self-sustained sequencereplication (Guatelli et al., (1989) Proc. Nat. Acad. Sci. 87:1874), andnucleic acid based sequence amplification (NABSA), or any other nucleicacid amplification method, followed by the detection of the amplifiedmolecules using techniques well known to those of skill in the art.These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

In one embodiment, any of a variety of sequencing reactions known in theart can be used to directly sequence at least a portion of an Fndc5- oririsin-encoding gene, or portion thereof, and detect allelic variants,e.g., mutations, by comparing the sequence of the sample sequence withthe corresponding reference (control) sequence. Exemplary sequencingreactions include those based on techniques developed by Maxam andGilbert (Proc. Natl Acad Sci USA (1977) 74:560) or Sanger (Sanger et al.(1977) Proc. Nat. Acad. Sci. 74:5463). It is also contemplated that anyof a variety of automated sequencing procedures may be utilized whenperforming the subject assays (Biotechniques (1995) 19:448), includingsequencing by mass spectrometry (see, for example, U.S. Pat. No.5,547,835 and international patent application Publication Number WO94/16101, entitled DNA Sequencing by Mass Spectrometry by H. Köster;U.S. Pat. No. 5,547,835 and international patent application PublicationNumber WO 94/21822 entitled “DNA Sequencing by Mass Spectrometry ViaExonuclease Degradation” by H. Köster), and U.S. Pat. No. 5,605,798 andInternational Patent Application No. PCT/US96/03651 entitled DNADiagnostic's Based on Mass Spectrometry by H. Köster; Cohen et al.(1996) Adv Chromatogr 36:127-162; and Griffin et al. (1993) Appl BiochemBiotechnol 38:147-159). It will be evident to one skilled in the artthat, for certain embodiments, the occurrence of only one, two or threeof the nucleic acid bases need be determined in the sequencing reaction.For instance, A-track or the like, e.g., where only one nucleotide isdetected, can be carried out.

Yet other sequencing methods are disclosed, e.g., in U.S. Pat. No.5,580,732 entitled “Method of DNA sequencing employing a mixedDNA-polymer chain probe” and U.S. Pat. No. 5,571.676 entitled “Methodfor mismatch-directed in vitro DNA sequencing”.

In some cases, the presence of a specific allele of an Fndc5- oririsin-encoding gene in DNA from a subject can be shown by restrictionenzyme analysis. For example, a specific nucleotide polymorphism canresult in a nucleotide sequence comprising a restriction site which isabsent from the nucleotide sequence of another allelic variant.

In a further embodiment, protection from cleavage agents (such as anuclease, hydroxylamine or osmium tetroxide and with piperidine) can beused to detect mismatched bases in RNA/RNA DNA/DNA, or RNA/DNAheteroduplexes (Myers, et al. (1985) Science 230:1242). In general, thetechnique of “mismatch cleavage” starts by providing heteroduplexesformed by hybridizing a control, nucleic acid, which is optionallylabeled, e.g., RNA or DNA, comprising a nucleotide sequence of an PGC-1αallelic variant with a sample nucleic acid, e.g., RNA or DNA, obtainedfrom a tissue sample. The double-stranded duplexes are treated with anagent which cleaves single-stranded regions of the duplex such asduplexes formed based on basepair mismatches between the control andsample strands. For instance, RNA/DNA duplexes can be treated with RNaseand DNA/DNA hybrids treated with S1 nuclease to enzymatically digest themismatched regions. In other embodiments, either DNA/DNA or RNA/DNAduplexes can be treated with hydroxylamine or osmium tetroxide and withpiperidine in order to digest mismatched regions. After digestion of themismatched regions, the resulting material is then separated by size ondenaturing, polyacrylamide gels to determine whether the control andsample nucleic acids have an identical nucleotide sequence or in whichnucleotides they are different. See, for example, Cotton et al. (1988)Proc. Acad Sci USA 85:4397 Saleeba et al. (1992) Methods Enzymol.217:286-295. In a preferred embodiment, the control or sample nucleicacid is labeled for detection.

In another embodiment, an allelic variant can be identified bydenaturing high-performance liquid chromatography (DHPLC) (Oefner andUnderhill, (1995) Am. J. Human Gen. 57: Suppl. A266). DHPLC usesreverse-phase ion-pairing chromatography to detect the heteroduplexesthat are generated during amplification of PCR fragments fromindividuals who are heterozygous at a particular nucleotide locus withinthat fragment (Oefner and Underhill (1995) Am. J. Human Gen. 57: Suppl.A266). In general, PCR products are produced using PCR primers flankingthe DNA of interest. DHPLC analysis is carried out and the resultingchromatograms are analyzed to identify base pair alterations ordeletions based on specific chromatographic profiles (see O'Donovan etal. (1998) Genomics 52:44-49).

In other embodiments, alterations in electrophoretic mobility is used toidentify the type of desired allelic variant. For example, single strandconformation polymorphism (SSCP) may be used to detect differences inelectrophoretic mobility between mutant and wild type nucleic acids(Orita et al. (1989) Proc Natl. Acad. Sci USA 86:2766: see also Cotton(1993) Mutat Res 285:125-144; and Hayashi (1992) Genet Anal Tech. Appl9:73-79). Single-stranded DNA fragments of sample and control nucleicacids are denatured and allowed to renature. The secondary structure ofsingle-stranded nucleic acids varies according to sequence, theresulting alteration in electrophoretic mobility enables the detectionof even a single base change. The DNA fragments may be labeled ordetected with labeled probes. The sensitivity of the assay may beenhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In anotherpreferred embodiment, the subject method utilizes heteroduplex analysisto separate double stranded heteroduplex molecules on the basis ofchanges in electrophoretic mobility (Keen et al. (1991) Trends Genet7:5).

In yet another embodiment, the identity of an allelic variant of apolymorphic region is obtained by analyzing the movement of a nucleicacid comprising the polymorphic region in polyacrylamide gels containinga gradient of denaturant is assayed using denaturing gradient gelelectrophoresis (DGGE) (Myers et al. (1985) Nature 313:495). When DGGEis used as the method of analysis, DNA will be modified to insure thatit does not completely denature, for example by adding a GC clamp ofapproximately 40 bp of high-melting GC-rich DNA by PCR. In a furtherembodiment, a temperature gradient is used in place of a denaturingagent gradient to identify differences in the mobility of control andsample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:1275).

Examples of techniques for detecting differences of at least onenucleotide between two nucleic acids include, but are not limited to,selective oligonucleotide hybridization, selective amplification, orselective primer extension. For example, oligonucleotide probes may beprepared in which the known polymorphic nucleotide is placed centrally(allele-specific probes) and then hybridized to target DNA underconditions which permit hybridization only if a perfect match is found(Saiki et. al. (1986) Nature 324:163); Saiki et al (1989) Proc. NatlAcad. Sci USA 86:6230: and Wallace et al. (1979) Nucl. Acids Res.6:3543). Such allele specific oligonucleotide hybridization techniquesmay be used for the simultaneous detection of several nucleotide changesin different polylmorphic regions of Fndc5- or irisin-encoding genes.For example, oligonucleotides having nucleotide sequences of specificallelic variants are attached to a hybridizing membrane and thismembrane is then hybridized with labeled sample nucleic acid. Analysisof the hybridization signal will then reveal the identity of thenucleotides of the sample nucleic acid.

Alternatively, allele specific amplification technology which depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the allelic variant of interest in the center of the molecule(so that amplification depends on differential hybridization) (Gibbs etal. (1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end ofone primer where, under appropriate conditions, mismatch can prevent, orreduce polymerase extension (Prossner (1993) Tibtech 11:238, Newton etal. (198.9) Nucl. Acids Res. 17:2503). This technique is also termed“PROBE” for Probe Oligo Base Extension. In addition, it may be desirableto introduce a novel restriction site in the region of the mutation tocreate cleavage-based detection (Gasparini et al. (1992) Mol Cell Probes6:1).

In another embodiment, identification of the allelic variant is carriedout using an oligonucleotide ligation assay (OLA), as described, e.g.,in U.S. Pat. No. 4,998,617 and Landegren, U. et al., (1988) Science241:1077-1080. The OLA protocol uses two oligonucleotides which aredesigned to be capable of hybridizing to abutting sequences of a singlestrand of a target. One of the oligonucleotides is linked to aseparation marker, e.g., biotinylated, and the other is detectablylabeled. If the precise complementary sequence is found in a targetmolecule, the oligonucleotides will hybridize such that their terminiabut, and create a ligation substrate. Ligation then permits the labeledoligonucleotide to be recovered using avidin, or another biotin ligand.Nickerson, D. A. et al. have described a nucleic acid detection assaythat combines attributes of PCR and OLA (Nickerson, D. A. et al., (1990)Proc. Natl. Acad. Sci. (U.S.A.) 87:8923-8927. In this method, PCR isused to achieve the exponential amplification of target DNA, which isthen detected using OLA.

Several techniques based on this OLA method have been developed and canbe used to detect specific allelic variants of a polymorphic region ofan PGC-1α gene. For example, U.S. Pat. No. 5,593,826 discloses an OLAusing an oligonucleotide having 3′-amino group and a 5′-phosphorylatedoligonucleotide to form a conjugate having a phosphoramidate linkage. Inanother variation of OLA described in Tobe et al. ((1996) Nucleic AcidsRes 24: 3728), OLA combined with PCR permits typing of two alleles in asingle microtiter well. By marking each of the allele-specific primerswith a unique hapten, i.e. digoxigenin and fluorescein, each OLAreaction can be detected by using hapten specific antibodies that arelabeled with different enzyme reporters, alkaline phosphatase orhorseradish peroxidase. This system permits the detection of the twoalleles using a high throughput format that leads to the production oftwo different colors.

The invention further provides methods for detecting single nucleotidepolymorphisms in an Fndc5- or irisin-encoding gene. Because singlenucleotide polymorphisms constitute sites of variation flanked byregions of invariant sequence, their analysis requires no more than thedetermination of the identity of the single nucleotide present at thesite of variation and it is unnecessary to determine a complete genesequence for each subject. Several methods have been developed tofacilitate the analysis of such single nucleotide polymorphisms.

In one embodiment, the single base polymorphism can be detected by usinga specialized exonuclease-resistant nucleotide, as disclosed, in Mundy,C. R. (U.S. Pat. No. 4,656,127). According to the method, a primercomplementary to the allelic sequence immediately 3′ to the polymorphicsite is permitted to hybridize to a target molecule obtained from aparticular animal or human. If the polymorphic site on the targetmolecule contains a nucleotide that is complementary to the particularexonuclease-resistant nucleotide derivative present, then thatderivative will be incorporated onto the end of the hybridized primer.Such incorporation renders the primer resistant to exonuclease, andthereby permits its detection. Since the identity of theexonuclease-resistant derivative of the sample is known, a finding thatthe primer has become resistant to exonucleases reveals that thenucleotide presents in the polymorphic site of the target molecule wascomplementary to that of the nucleotide derivative used in the reaction.This method has the advantage that it does not require the determinationof large amounts of extraneous sequence data.

In another embodiment of the invention, a solution-based method is usedfor determining the identity of the nucleotide of a polymorphic site(Cohen, D. et al. (French Patent 2,650,840; PCT Application No.WO91/02087). As in the Mundy method of U.S. Pat. No. 4,656,127, a primeris employed that is complementary to allelic sequences immediately 3′ toa polymorphic site. The method determines the identity of the nucleotideof that site using labeled dideoxynucleotide derivatives, which, ifcomplementary to the nucleotide of the polymorphic site will becomeincorporated onto the terminus of the primer.

An alternative method, known as Genetic Bit Analysis or GBA is describedby Goelet, P. et al. (PCT Application No. 92/15712). The method ofGoelet, P. et al. uses mixtures of labeled terminators and a primer thatis complementary to the sequence 3′ to a polymorphic site. The labeledterminator that is incorporated is thus determined by, and complementaryto, the nucleotide present in the polymorphic site of the targetmolecule being evaluated. In contrast to the method of Cohen et al.(French Patent 2,650,840; PCT Appln. No. WO91/02087) the method ofGoelet. P. et al. is preferably a heterogeneous phase assay, in whichthe primer or the target molecule is immobilized to a solid phase.

Several primer-guided nucleotide incorporation procedures for assayingpolymorphic sites in DNA have been described (Komher, J. S. et al.,Nucl. Acids. Res. 17:7779-7784 (1989), Sokolov, B. P., Nucl. Acids Res.18:3671 (1990), Syvanen, A.-C., et al., Genomics 8:684-692 (1990);Kuppuswamy, M. N. et al., Proc. Natl. Acad. Sci. (U.S.A.) 88:1143-1147(1991); Prezant, T. R. et al., Hum. Mutat. 1:159-164 (1992); Ugozzoli,L. et al., GATA 9:107-112 (1992); Nyren, P. et al., Anal. Biochem.208:171-175 (1993)). These methods differ from GBA in that they all relyon the incorporation of labeled deoxynucleotides to discriminate betweenbases at a polymorphic site. In such a format, since the signal isproportional to the number of deoxynucleotides incorporated,polymorphisms that occur in runs of the same nucleotide can result insignals that are proportional to the length of the run (Syvanen, A.-C.,et al., Amer. J. Hum. Genet. 52:46-59 (1993)).

For determining the identity of the allelic variant of as polymorphicregion located in the coding region of an Fndc5- or irisin-encodinggene, yet other methods than those described above can be used. Forexample, identification of an allelic variant which encodes a mutatedprotein can be performed by using an antibody specifically recognizingthe mutant protein in, e.g., immunohistochemistry orimmunoprecipitation. Antibodies to wild-type Fndc5 or irisin, orfragment thereof, or mutated forms of such proteins can be preparedaccording to methods known in the art.

Alternatively, one can also measure an activity of a BDNF or Fndc5 oririsin polypeptide, or fragments thereof, such as the ability to crossthe blood-brain barrier, to enhance BDNF expression, to bind to neurons,and the like. Binding assays are known in the art and involve, e.g.,obtaining cells from a subject, and performing binding experiments witha labeled lipid, to determine whether binding to the mutated form of theprotein differs from binding to the wild-type of the protein.

Antibodies directed against reference or mutant BDNF or Fndc5 or irisinpolypeptides, or fragments thereof can also be used in diseasediagnostics and prognostics. Such antibodies are well known in the art(see, for example, antibody LS-C166197 from Lifespan Biosciences,antibody AG-25B-0027 from Adipogen, antibody HPA051290 from AtlasAntibodies, antibody PAN576Hu02 from Uscn Lifesciences, antibodyAP18024PU-N from Acris Antibodies, antibody OAAB05345 from Aviva SystemsBiology, antibody CPBT-33932RH from Creative Biomart, antibody Orb39441from Biorbyt, and antibody NBP2-14024 from Novus Biologicals). Inaddition, such diagnostic methods, may be used to detect abnormalitiesin the level of such polypeptide expression, or abnormalities in thestructure and/or tissue, cellular, or subcellular location of suchpolypeptides. Structural differences may include, for example,differences in the size, electronegativity, or antigenicity of themutant polypeptide relative to the normal polypeptide. Protein from thetissue or cell type to be analyzed may easily be detected or isolatedusing techniques which are well known to one of skill in the art,including but not limited to Western blot analysis. For a detailedexplanation of methods for carrying out Western blot analysis, seeSambrook et al. 1989. supra, at Chapter 18. The protein detection andisolation methods employed herein may also be such as those described inHarlow and Lane, for example (Harlow, E. and Lane, D., 1988,“Antibodies: A Laboratory Manual”, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.), which is incorporated herein by reference inits entirety.

This can be accomplished, for example, by immunofluorescence techniquesemploying a fluorescently labeled antibody (see below) coupled withlight microscopic, flow cytometric, or fluorimetric detection. Theantibodies (or fragments thereof) useful in the present invention may,additionally, be employed histologically, as in immunofluorescence orimmunoelectron microscopy, for in situ detection of Fndc5 or irisinpolypeptide, or fragments thereof. In situ detection may be accomplishedby removing a histological specimen from a subject, and applying theretoa labeled antibody of the present invention. The antibody (or fragment)is preferably applied by overlaying the labeled antibody (or fragment)onto a biological sample. Through the use of such a procedure, it ispossible to determine not only the presence of the Fndc5 or irisinpolypeptide, or fragments thereof, but also its distribution in theexamined tissue. Using the present invention, one of ordinary skill willreadily perceive that any of a wide variety of histological methods(such as staining procedures) can be modified in order to achieve suchin situ detection.

Often a solid phase support or carrier is used as a support capable ofbinding an antigen or an antibody. Well-known supports or carriersinclude glass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, gabbros, andmagnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material may have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding toan antigen or antibody. Thus, the support configuration may bespherical, as in a bead, or cylindrical, as in the inside surface of atest tube, or the external surface of a rod. Alternatively, the surfacemay be flat such as a sheet, test strip, etc. Preferred supports includepolystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

One means for labeling an antibody is via linkage to an enzyme and usein an enzyme immunoassay (EIA) (Voller, “The Enzyme Linked ImmunosorbentAssay (ELISA)”, Diagnostic Horizons 2:1-7, 1978, MicrobiologicalAssociates Quarterly Publication, Walkersville, Md.; Voller, et al., J.Clin. Pathol. 31:507-520 (1978); Butler, Meth. Enzymol. 73:482-523(1981); Maggio, (ed.) Enzyme Immunoassay, CRC Press Boca Raton, Fla.,1980; Ishikawa, et al., (eds.) Enzyme Immunoassay, Kgaku Shoin, Tokyo,1981). The enzyme which is bound to the antibody will react with anappropriate substrate, preferably a chromogenic substrate, in such amanner as to produce a chemical moiety which can be detected, forexample, by spectrophotometric, fluorimetric or by visual means. Enzymeswhich can be used detectably label the antibody include, but are notlimited to, malate dehydrogenase, staphylococcal nuclease,delta-5-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate, dehydrogenase, those phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. The detection can be accomplished by colorimetricmethods which employ a chromogenic substrate for the enzyme. Detectionmay also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards.

Detection may also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the antibodies orantibody fragments, it is possible to detect fingerprint gene wild typeor mutant peptides through the use of a radioimmunoassay (RIA) (see, forexample, Weintraub, B., Principles of Radioimmunoassays, SeventhTraining Course on Radioligand Assay Techniques, The Endocrine Society,March 1986, which is incorporated by reference herein). The radioactiveisotope can be detected by such means as the use of a gamma counter or ascintillation counter or by autoradiography.

It is also possible to label the antibody with a fluorescent compound.When the fluorescently labeled antibody is exposed to light of theproper wave length, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine. Theantibody can also be detectably labeled using fluorescence emittingmetals such as ¹⁵²Eu, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the Chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminal, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody ofthe present invention. Bioluminescence is a type of chemiluminescencefound in biological systems in, which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, luciferase and aequorin.

If a polymorphic region is located in an exon, either in a coding ornon-coding portion of the gene, the identity of the allelic variant canbe determined by determining the molecular structure of the mRNA,pre-mRNA, or cDNA. The molecular structure can be determined using anyof the above described methods for determining the molecular structureof the genomic DNA.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits, such as those described above, comprisingat least one probe or primer nucleic acid described herein, which may beconveniently used, e.g., to determine whether a subject has or is atrisk of developing a disease associated with a specific allelic variantof interest. Sample nucleic acid to be analyzed by any of theabove-described diagnostic and prognostic methods can be obtained fromany cell type or tissue of a subject. For example, a subject's bodilyfluid (e.g. blood) can be obtained by known techniques (e.g.,venipuncture). Alternatively, nucleic acid tests can be performed on drysamples (e.g., hair or skin). Fetal nucleic acid samples can be obtainedfrom maternal blood as described in International Patent Application No.WO91/07660 to Bianchi. Alternatively, amniocytes or chorionic villi maybe obtained for performing prenatal testing.

Diagnostic procedures may also be performed in situ directly upon tissuesections (fixed and/or frozen) of subject tissue obtained from biopsiesor resections, such that no nucleic acid purification is necessary.Nucleic acid reagents may be used as probes and/or primers for such insitu procedures (see, for example, Nuovo, G. J., 1992, PCR in situhybridization: protocols and applications, Raven Press, NY).

In addition to methods which focus primarily on the detection of onenucleic acid sequence, profiles may also be assessed in such detectionschemes. Fingerprint profiles may be generated, for example, byutilizing a differential display procedure, Northern analysis and/orRT-PCR.

B. Monitoring of Effects During Clinical Trials

The present invention further provides methods for determining theeffectiveness of a BDNF modulator (e.g., an FNDC5 polypeptide/nucleicacid, or fragment thereof) in treating or preventing a neurologicaldisease or disorder or assessing risk of developing a neurologicaldisease or disorder in a subject. For example, the effectiveness of sucha modulator in increasing BDNF gene expression, protein levels can bemonitored in clinical trials of subjects. In such clinical trials, theexpression or activity of an Fndc5 gene, BDNF gene, or other genes thathave been implicated in, for example, a BDNF expression pathway can beused as a “read out” or marker of the phenotype of a particular cell.

For example, and not by way of limitation, genes, including BDNF, thatare modulated in cells by treatment with an agent that modulates Fndc5expression or activity can be identified. Thus, to study the effect ofagents which modulate BDNF expression or activity in subjects sufferingfrom or at risk of developing a neurological disease or disorder, oragents to be used as a prophylactic, for example, a clinical trial,cells can be isolated and RNA prepared and analyzed for the levels ofexpression of BDNF and other genes implicated in BDNF activity orexpression. The levels of gene expression (e.g., a gene expressionpattern) can be quantified by Northern blot analysis or RT-PCR, asdescribed herein, or alternatively by measuring the amount of proteinproduced, by one of the methods described herein, or by measuring thelevels of activity of BDNF or other genes, such as the BDNF regulator,Npas4. In this way, the gene expression pattern can serve as a marker,indicative of the physiological response of the cells to the agent whichmodulates BDNF expression or activity. This response state may bedetermined before, and at various points during treatment of theindividual with the agent which modulates BDNF expression or activity

In a preferred embodiment, the present invention provides a method formonitoring the effectiveness of treatment of a subject with an agentwhich modulates BDNF expression or activity (e.g., an agonist,antagonist, peptidomimetic, protein, peptide, nucleic acid, siRNA, orsmall molecule identified by the screening assays described herein)including the steps of (i) obtaining a pre-administration simple from asubject prior to administration of the agent, preferably a sample fromthe central or peripheral nervous system; (ii) detecting the level ofexpression of a BDNF protein, mRNA, or genomic DNA in thepre-administration sample; (iii) obtaining, one or morepost-administration samples from the subject; (iv) detecting the levelof expression or activity of the BDNF protein, mRNA, or genomic DNA inthe post-administration samples; (v) comparing the level of expressionor activity of the BDNF protein, mRNA, or genomic DNA in thepre-administration sample with the BDNF protein, mRNA, or genomic DNA inthe post administration sample or samples; and (vi) altering theadministration of the agent to the subject accordingly. For example,increased administration of the agent may be desirable to increase theexpression or activity of BDNF to higher levels than detected, i.e., toincrease the effectiveness of the agent. According to such anembodiment, BDNF expression or activity may be used as an indicator ofthe effectiveness of an agent, even in the absence of an observablephenotypic response,

IV. Isolated Nucleic Acids, Polypeptides, Antibodies, Vectors, and HostCells Useful for the Methods Described Herein

Nucleic acids, polypeptides, vectors, and host cells related to Fndc5 oririsin, or fragments thereof, are useful for carrying out the methodsdescribed herein.

Isolated nucleic acid molecules that encode Fndc5 or irisin, orbiologically alive portions thereof, are well known in the art. As usedherein, the term “nucleic acid molecule” is intended to include DNAmolecules cDNA or genomic DNA) and RNA molecules (i.e., mRNA) andanalogs of the DNA or RNA generated using nucleotide analogs. Thenucleic acid molecule can be single-stranded or double-stranded, butpreferably is double-stranded DNA. An “isolated” nucleic acid moleculeis one which is separated from other nucleic acid molecules which arepresent in the natural source of the nucleic acid. Preferably, an“isolated” nucleic acid is free of sequences which naturally flank thenucleic acid (i.e., sequences located at the 5′ and 3′ ends of thenucleic acid) in the genomic DNA of the organism from which the nucleicacid is derived. For example, in various embodiments, the isolated Fndc5nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flankthe nucleic acid molecule in genomic DNA of the cell from which thenucleic acid is derived (i.e., a brown adipocyte). Moreover, an“isolated” nucleic acid molecule, such as a cDNA molecule, can besubstantially free of other cellular material, or culture medium whenproduced by recombinant techniques, or chemical precursors or otherchemicals when chemically synthesized.

A nucleic acid molecule of the present invention, e.g. a nucleic acidmolecule having, the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9,11, 13 and 15 or a nucleotide sequence which is at least about 50%,preferably at least about 60%, more preferably at least about 70%, yetmore preferably at least about 80%, still more preferably at least about90%, and most preferably at least about 95% or more (e.g., about 98%)homologous to the nucleotide sequence shown in SEQ ID NOs: 1, 3, 5, 7,9, 11, 13 and 15 or a portion thereof (i.e., 100, 200, 300, 400, 450,500, or more nucleotides), can be isolated using standard molecularbiology techniques and the sequence information provided herein. Forexample, a human Fndc5 cDNA can be isolated from a human muscle cellline (from Stratagene, LaJolla, Calif., or Clontech, Palo Alto, Calif.)using all or portion of SEQ ID NOs: 1, 3, 5. 7, 9, 11, 13 or 15, orfragment thereof, as a hybridization probe and standard hybridizationtechniques (i.e., as described in Sambrook, Fritsh, E. F., and Maniatis,T. Molecular Cloning: A Laboratory Manual, 2nd, ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989). Moreover, a nucleic acid molecule encompassing or a portionof SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13 or 15 or a nucleotide sequencewhich is at least about 50%, preferably at least about 60%, morepreferably at least about 70%, yet more preferably at least about 80%,still more preferably at least about 90%, and most preferably at leastabout 95% or more homologous to the nucleotide sequence shown in SEQNOs: 1, 3, 5, 7, 9, 11, 13 or 15, or fragment thereof, can be isolatedby the polymerase chain reaction using oligonucleotide primers designedbased upon the sequence of SEQ NOs: 1, 3, 5, 7, 9, 11, 13 or 15, orfragment thereof, or the homologous nucleotide sequence. For example,mRNA can be isolated from muscle cells (i.e., by theguanidinium-thiocyanate extraction procedure of Chirgwin et al. (1979)Biochemistry 18: 5294-5299) and cDNA can be prepared using reversetranscriptase (i.e., Moloney MLV reverse transcriptase, available fromGibco/BRL, Bethesda, Md.: or AMV reverse transcriptase, available fromSeikagaku America, Inc., St. Petersburg, Fla.). Syntheticoligonucleotide primers for PCR amplification can be designed based uponthe nucleotide sequence shown in SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13 or15, or fragment thereof, or to the homologous nucleotide sequence. Anucleic acid of the invention can be amplified using cDNA or,alternatively, genomic DNA, as a template and appropriateoligonucleotide primers according to standard PCR amplificationtechniques. The nucleic acid so amplified can be cloned into anappropriate vector and characterized by DNA sequence analysis.Furthermore, oligonucleotides corresponding to an Fndc5 nucleotidesequence can be prepared by standard synthetic techniques, i.e., usingan automated DNA synthesizer.

Probes based on the Fndc5 nucleotide sequences can be used to detecttranscripts or genomic sequences encoding the same or homologousproteins. In preferred embodiments, the probe further comprises a labelgroup attached thereto, i.e., the label group can be a radioisotope, afluorescent compound, an enzyme, or an enzyme co-factor. Such probes canbe used as a part of a diagnostic test kit for identifying cells ortissue which express an Fndc5 protein, such as by measuring, a level ofan Fndc5-encoding nucleic acid in a sample of cells from a subject,i.e., detecting Fndc5 mRNA levels.

Nucleic acid molecules encoding other Fndc5 members and thus which havea nucleotide sequence which differs from the Fndc5 sequences of SEQ IDNOs: 1, 3, 5, 7, 9, 11, 13 or 15, or fragment thereof, are contemplated.Moreover, nucleic acid molecules encoding Fndc5 proteins from differentspecies, and thus which have a nucleotide sequence which differs fromthe Fndc5 sequences of SEQ ID NOs: 1, 3 5, 7, 9, 11, 13 or 15 are alsointended to be within the scope of the present invention. For example,rat or monkey Fndc5 cDNA can be identified based on the nucleotidesequence of a human and/or mouse Fndc5.

In one embodiment, the nucleic acid molecule(s) of the invention encodesa protein or portion thereof which includes an amino acid sequence whichis sufficiently homologous to an amino acid sequence of SEQ ID NO: 2, 4,6, 8, 10, 12 or 14, or fragment thereof, such that the protein orportion thereof modulates (e.g., enhance) one or more of the followingbiological activities: 1) BDNF expression in the central and/orperipheral nervous system; 2) activity-induced immediate-early geneexpression in neurons; 3) neuronal survival; 4) neurological lesionformation; 5) neurite outgrowth; 6) synaptogenesis; 7) synapticplasticity; 8) neuronal mitochondrial function; 9) dendriticarborization; 10) neuronal differentiation; and 11) neuronal migration.

As used herein, the language “sufficiently homologous” refers toproteins or portions thereof which have amino acid sequences whichinclude a minimum number of identical or equivalent (e.g., an amino acidresidue which has a similar side chain as an amino acid residue in SEQID NO: 2, 4, 6, 8, 10, 12 or 14, or fragment thereof) amino acidresidues to an amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12 or14, or fragment thereof, such that the protein or portion thereofmodulates (e.g., enhance) one or more of the following biologicalactivities: 1) BDNF expression in the central and/or peripheral nervoussystem; 2) activity-induced immediate-early gene expression in neurons;3) neuronal survival; 4) neurological lesion formation; 5) neuriteoutgrowth; 6) synaptogenesis; 7) synaptic, plasticity; 8) neuronalmitochondrial function; 9) dendritic arborization; 10) neuronaldifferentiation; and 11) neuronal migration.

In another embodiment, the protein is at least about 50%, re tenthly atleast about 60%, more preferably at least about 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to theentire amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12 or 14, orfragment thereof, or a frament thereof.

Portions of proteins encoded by Fndc5 or irisin nucleic acid moleculesare preferably biologically active portions of the Flide5 or irisinprotein. As used herein, the term “biologically active portion” isintended to include a portion, e.g., a domain/motif, of Fndc5 or irisinthat has one or more of the biological activities of the full-lengthFndc5 or irisin protein.

Standard binding assays, e.g, inununoprecipitations and yeast two-hybridassays, as described herein, or functional assays, RNAi oroverexpression experiments, can be performed to determine the ability ofan Fndc5 or irisin protein or a biologically active fragment thereof tomaintain a biological activity of the full-length Fndc5 or irisinprotein.

The invention further encompasses nucleic acid molecules that differfrom the nucleotide sequence shown in SEO ID NO: 1, 3, 5, 7, 9, 11, 13or 15, or fragment thereof due to degeneracy of the genetic code andthus encode the same Fndc5 or irisin protein as that encoded by thenucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15, forfragment thereof. In another embodiment, an isolated nucleic acidmolecule of the invention has a nucleotide sequence encoding a proteinhaving an amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8, 10, 12 or14, or fragment thereof, or fragment thereof, or a protein having anamino acid sequence which is at least about 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more homologous to theamino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12 or 14, or fragmentthereof, or a fragment thereof, or differs by at least 1, 2, 3, 5 or 10amino acids but not more than 30, 20, 15 amino acids from SEQ ID NO: 2,4, 6, 8, 10, 12 or 14. In another embodiment, a nucleic acid encoding anFndc5 or irisin polypeptide consists of nucleic acid sequence encoding aportion of a full-length Fndc5 or irisin fragment of interest that isless than 195, 190, 185, 180, 175, 170, 165, 160, 155, 150, 145, 140,135, 130, 125, 120, 115, 110, 105, 100, 95, 90, 85, 80, 75, or 70 aminoacids in length.

It will be appreciated by those skilled in the art that DNA sequencepolymorphisms that lead to changes in the amino acid sequences of Fndc5or irisin may exist within a population (e.g., a mammalian population, ahuman population). Such genetic polymorphism in the Fndc5 gene existamong individuals within a population due to natural allelic variation.As used herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules comprising an open reading frame encoding Fndc5 or irisinprotein, preferably a mammalian, e.g., human, Fndc5 or irisin protein.Such natural variations can typically result in 1-5% variance in thenucleotide sequence of the Fndc5 gene. Any and all such nucleotidevariations and resulting amino acid polymorphisms in Fndc5 that are theresult of natural allelic variation and that do not alter the functionalactivity of Fndc5 or irisin are intended to be within the scope of theinvention. Moreover, nucleic acid molecules encoding Fndc5 or irisinproteins from other species, and thus which have a nucleotide sequencewhich differs from the human or mouse sequences of SEQ ID NO: 1, 3, 5,or 7, are intended to be within the scope of the invention. Nucleic acidmolecules corresponding to natural allelic variants and homologues ofthe human or mouse Fndc5 cDNAs of the invention can be isolated based ontheir homology to the human or mouse Fndc5 nucleic acid sequencesdisclosed herein using the human or mouse cDNA, or a portion thereof, asa hybridization probe according to standard hybridization techniquesunder stringent hybridization conditions (as described herein).

In addition to naturally-occurring allelic variants of the Fndc5sequence that may exist in the population, the skilled artisan willfurther appreciate that changes can be introduced by mutation into thenucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15, orfragment thereof, thereby leading to changes in the amino acid sequenceof the encoded Fndc5 or irisin protein, without altering the functionalability of the Fndc5 or irisin protein. For example, nucleotidesubstitutions leading to amino acid substitutions at “non-essential”amino acid residues can be made in the sequence SEQ ID NO: 1, 3, 5, 7,9, 11, 13 or 15, or fragment thereof. A “non-essential” amino acidresidue is a residue that can be altered from the wild-type sequence ofFndc5 (e.g., the sequence of SEQ ID NO: 2, 6, 8, 10, 12 or 14, orfragment thereof) without altering the activity of Fndc5 or irisin,whereas an “essential” amino acid residue is required for Fndc5 oririsin activity. Other amino acid residues, however, (e.g., those thatare not conserved or only semi-conserved between mouse and human) maynot be essential for activity and thus are likely to be amenable toalteration without altering Fndc5 or irisin activity. Furthermore, aminoacid residues that are essential for Fndc5 or irisin functions relatedto neurological disorders, but not essential for Fndc5 functions relatedto thermogenesis, gluconeogenesis, cellular metabolism, and the like arelikely to be amenable to alteration.

Accordingly, another aspect of the invention pertains to nucleic acidmolecules encoding Fndc5 or irisin proteins that contain changes inamino acid residues that are not essential for Fndc5 or irisin activity.Such Fndc5 or irisin proteins differ in amino acid sequence from SEQ IDNO: 2, 4, 6, 8, 10, 12 or 14, or fragment thereof yet retain at leastone of the Fndc5 or irisin activities described herein. In oneembodiment, the isolated nucleic acid molecule comprises a nucleotidesequence encoding a protein, wherein the protein lacks one or more Fndc5or irisin domains (e.g., a fibronectin, extracellular, signal peptide,hydrophobic, and/or C-terminal domain).

“Sequence identity or homology”, as used herein, refers to the sequencesimilarity between two polypeptide molecules or between two nucleic acidmolecules. When a position in both of the two compared sequences isoccupied by the same base or amino acid monomer subunit, e.g., if aposition in each of two DNA molecules is occupied by adenine, then themolecules are homologous or sequence identical at that position. Thepercent of homology or sequence identity between two sequences is afunction of the number of matching or homologous identical positionsshared by the two sequences divided by the number of positionscompared×100, For example, if 6 of 10, of the positions in two sequencesare the same then the two sequences are 60% homologous or have 60%sequence identity. By way of example, the DNA sequences ATTGCC andTATGGC share 50% homology or sequence identity. Generally, a comparisonis made when two sequences arc aligned to give maximum homology. Unlessotherwise specified “loop out regions”, e.g., those arising from, fromdeletions or insertions in one of the sequences are counted asmismatches.

The comparison of sequences and determination of percent homologybetween two sequences can be accomplished using a mathematicalalgorithm. Preferably, the alignment can be performed using the ClustalMethod. Multiple alignment parameters include GAP Penalty=10, Gap LengthPenalty=10. For DNA alignments, the pairwise alignment parameters can beHtuple=2Gap penalty=5, Window=4, and Diagonal saved=4. For proteinalignments, the pairwise alignment parameters can be Ktuple=1, Gappenalty=3, Window=5, and Diagonals Saved=5.

In a preferred embodiment, the percent identity between two amino acidsequences is determined using the Needleman and Wunsch (J. Mol. Biol.(48):444-453 (1970)) algorithm which has been incorporated into the GAPprogram in the GCG software package (available online), using either aBlossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12,10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6, In yetanother preferred embodiment, the percent identity between twonucleotide sequences is determined using the GAP program in the GCGsoftware package (available online), using a NWSgapdna.CMP matrix and agap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4,5, or 6. In another embodiment, the percent identity between two aminoacid or nucleotide sequences is determined using the algorithm of E.Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has beenincorporated into the ALIGN program (version 2.0) (available online),using a PAM 120 weight residue table, a gap length penalty of 12 and agap penalty of 4.

An isolated nucleic acid molecule encoding an Fndc5 or irisin proteinhomologous to the protein of SEQ ID NO: 2, 4, 6, 8, 10, 12 or 14, orfragment thereof, can be created by introducing one or more nucleotidesubstitutions, additions or deletions into the nucleotide sequence ofSEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15, or fragment thereof, or ahomologous nucleotide sequence such that one or more amino acidsubstitutions, additions or deletions are introduced into the encodedprotein. Mutations can be introduced into SEQ ID NO: 1, 3, 5, 7, 9, 11,13 or 15, or fragment thereof, or the homologous nucleotide sequence bystandard techniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Preferably, conservative amino acid substitutions are madeat one or more predicted non-essential amino acid residues. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alimine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), bet217-420ranched sidechains e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan histidine). Thus, a predictednonessential amino acid residue in Fndc5 or irisin is preferablyreplaced with another amino acid residue from the same side chainfamily. Alternatively, in another embodiment, mutations can beintroduced randomly along all or part of an Fndc5 coding sequence, suchas by saturation mutagenesis, and the resultant mutants can be screenedfor an Fndc5 or irisin activity described herein to identify mutantsthat retain Fndc5 or irisin activity. Following mutagenesis of SEQ IDNO: 1, 3, 5, 7, 9, 11, 13 or 15, or fragment thereof, the encodedprotein can be expressed recombinantly (as described herein) and theactivity of the protein can be determined using, for example, assaysdescribed herein.

Fndc5 levels may be assessed by any of a wide variety of well knownmethods for detecting expression of a transcribed molecule or protein.Non-limiting examples of such methods include immunological methods fordetection of proteins, protein purification methods, protein function oractivity assays, nucleic acid hybridization methods, nucleic acidreverse transcription methods, and nucleic acid amplification methods.

In preferred embodiments, Fndc5 levels are ascertained by measuring genetranscript (e.g., mRNA), by a measure of the quantity of translatedprotein, or by a measure of gene product activity. Expression levels canbe monitored in a variety of ways, including by detecting mRNA levels,protein levels, or protein activity, any of which can be measured usingstandard techniques. Detection can involve quantification of the levelof gene expression (e.g., genomic DNA, cDNA, mRNA, protein, or enzymeactivity), or, alternatively, can be a qualitative assessment of thelevel of gene expression, in particular in comparison with a controllevel. The type of level being detected will be clear from the context.

In a particular embodiment, the Fndc5 mRNA expression level can bedetermined both by in situ and by in vitro formats in a biologicalsample using methods known in the art. The term “biological sample” isintended to include tissues, cells, biological fluids and isolatesthereof, isolated from a subject, as well as tissues, cells and fluidspresent within a subject. Many expression detection methods use isolatedRNA. For in vitro methods, any RNA isolation technique that does notselect against the isolation of mRNA can be utilized for thepurification of RNA from cells (see, e.g., Ausubel et al., ed., CurrentProtocols in Molecular Biology, John Wiley & Sons, New York 1987-1999).Additionally, large numbers of tissue samples can readily be processedusing techniques well known to lose of skill in the art, such as, forexample, the single-step RNA isolation process of Chomczynski (1989,U.S. Pat. No. 4,843,155).

The isolated mRNA can be used in hybridization or amplification assaysthat include, but are not limited to, Southern or Northern analyses,polymerase chain reaction analyses and probe arrays. One preferreddiagnostic method for the detection of mRNA levels involves contactingthe isolated mRNA with a nucleic acid molecule (probe) that canhybridize to the mRNA encoded by the gene being detected. The nucleicacid probe can be, for example, a full-length cDNA, or a portionthereof, such as an oligonucleotide of at least 7, 15, 30, 50, 100, 250or 500 nucleotides in length and sufficient to specifically hybridizeunder stringent conditions to a mRNA genomic DNA encoding Fndc5. Othersuitable probes for use in the diagnostic assays of the invention aredescribed herein. Hybridization of an mRNA with the probe indicates thatFndc5 is being expressed.

In one format, the mRNA is immobilized on a solid surface and contactedwith a probe, for example by running the isolated mRNA on an agarose geland transferring the mRNA from the gel to a membrane, such asnitrocellulose. In an alternative format, the probe(s) are immobilizedon a solid surface and the mRNA is contacted with the probe(s), forexample, in a gene chip array e.g., an Affymetrix™ gene chip array. Askilled artisan can readily adapt known mRNA detection methods for usein detecting the level of the Fndc5 mRNA expression levels.

An alternative method for determining the Fndc5 mRNA expression level ina sample involves the process of nucleic acid amplification, e.g., byrtPCR (the experimental embodiment set forth in Mullis, 1987, U.S. Pat.No. 4,683,202), ligase chain reaction (Barany, 1991, Proc. Natl. Acad.Sci. USA, 88:189-193), self sustained sequence replication (Guatelli etal., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptionalamplification system (Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA86:1173-1177), Q-Beta Replicase (Lizardi et al., 1988, Bio/Technology6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No.5,854,033) or any other nucleic acid amplification method, followed bythe detection of the amplified molecules using techniques well-known tothose of skill in the art. These detection schemes are especially usefulfor the detection of nucleic acid molecules if such molecules arepresent in very low numbers. As used herein, amplification primers aredefined as being a pair of nucleic acid molecules that can anneal to 5′or 3′ regions of a gene (plus and minus strands, respectively, orvice-versa) and contain a short region in between. In general,amplification primers are from about 10 to 30 nucleotides in length andflank a region from about 50 to 200 nucleotides in length. Underappropriate conditions and with appropriate reagents, such primerspermit the amplification of a nucleic acid molecule comprising thenucleotide sequence flanked by the primers.

For in situ methods, mRNA does not need to be isolated from the cellsprior to detection in such methods, a cell or tissue sample isprepared/processed using known histological methods. The sample is thenimmobilized on a support, typically a glass slide, and then contactedwith a probe that can hybridize to the Fndc5 mRNA.

As an alternative to making determinations based on the absolute Fndc5expression level, determinations may be based on the normalized Fndc5expression level. Expression levels are normalized by correcting theabsolute Fndc5 expression level by comparing its expression to theexpression of a non-Fndc5 gene, e.g., a housekeeping gene that isconstitutively expressed. Suitable genes for normalization includehousekeeping genes such as the actin gene, or epithelial cell-specificgenes. This normalization allows the comparison of the expression levelin one sample, e.g., a subject sample, to another sample, e.g., a normalsample, or between samples from different sources.

The level or activity of an Fndc5 or irisin protein can also be detectedand/or quantified by detecting or quantifying the expressed polypeptide.The Fndc5 or irisin polypeptide can be detected and quantified by any ofa number of means well known to those of skill in the art. These mayinclude analytic biochemical methods such as electrophoresis, capillaryelectrophoresis, high performance chromatography (HPLC), thin layerchromatography (TLC), hyperdiffusion chromatography, and the like, orvarious immunological methods such as fluid or gel precipitin reactions,immunodiffusion (single or double), immunoelectrophoresis,radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs),immunofluorescent assays, Western blotting, and the like. A skilledartisan can readily adapt known protein/antibody detection methods foruse in determining whether cells express Fndc5 or irisin, or fragmentsthereof.

Also provided are soluble, purified and/or isolated forms of Fndc5 oririsin, or fragments thereof. Hereinafter, irisin and fragments thereofwill be considered to be encompassed within the term “fragments ofFndc5.”

In one aspect, an Fndc5 polypeptide may comprise a full-length Fndc5amino acid sequence or a full-length Fndc5 amino acid sequence with 1 toabout 20 conservative amino acid substitutions. Amino acid sequence ofany Fndc5 polypeptide described herein can also be at least 50, 55, 60,65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 99.5%identical to an Fndc5 polypeptide sequence of interest, describedherein, well known in the art, or a fragment thereof. In addition, anyFndc5 polypeptide, or fragment thereof, described herein has modulates(e.g., enhance) one or more of the following biological activities: 1)BDNF expression in the central and/or peripheral nervous system; 2)activity-induced immediate-early gene expression in neurons; 3) neuronalsurvival; 4) neurological lesion formation; 5) neurite outgrowth; 6)synaptogenesis; 7) synaptic plasticity; 8) neuronal mitochondrialfunction; 9) dendritic arborization; 10) neuronal differentiation; and11) neuronal migration. In another aspect, the present inventioncontemplates a composition comprising an isolated Fndc5 polypeptide andless than about 25%, or alternatively 15%, of alternatively 5%,contaminatina bioloaical macromolecules of polypeptides.

The present invention further provides compositions related toproducing, detecting, or characterizing an Fndc5 polypeptide, orfragment thereof, such as nucleic acids, vectors, host cells, and thelike. Such compositions may serve as compounds that modulate an Fndc5polypeptide's expression and/or activity, such as antisense nucleicacids.

In certain embodiments, an Fndc5 polypeptide of the invention may be afusion protein containing a domain which increases its solubility andbioavailability and/or facilitates its purification, identification,detection, and/or structural characterization. Exemplary domains,include, for example, glutathione S-transferase (GST), protein A,protein G, calmodulin-binding peptide thioredoxin, maltose bindingprotein, HA, myc, poly arginine, poly His, poly His-Asp or FLAG fusionproteins and tags. Additional exemplary domains include domains thatalter protein localization in vivo, such as signal peptides, type IIIsecretion system-targeting peptides, transcytosis domains, nuclearlocalization signals, etc. In various embodiments, an Fndc5 polypeptideof the invention may comprise one or more heterologous fusions.Polypeptides may contain multiple copies of the same fusion domain ormay contain fusions to two or more different domains. The fusions mayoccur at the N-terminus of the polypeptide, at the C-terminus of thepolypeptide, or at both the N- and C-terminus of the polypeptide. It isalso within the scope of the invention to include linker sequencesbetween a polypeptide of the invention and the fusion domain in order tofacilitate construction of the fusion protein or to optimize proteinexpression or structural constraints of the fusion protein. In oneembodiment, the linker is a linker described herein, e.g., a linker ofat least 8, 9, 10, 15, 20 amino acids. The linker can be, e.g., anunstructured recombinant polymer (URP), e.g., a URP that is 9, 10, 11,12, 13, 14, 15, 20 amino acids in length, i.e., the linker has limitedor lacks secondary structure, e.g., Chou-Fasman algorithm. In anotherembodiment, the polypeptide may be constructed so as to contain proteasecleavage sites between the fusion polypeptide and polypeptide of theinvention in order to remove the tag after protein expression orthereafter. Examples of suitable endoproteases, include, for example,Factor Xa and TEV proteases.

In some embodiments, Fndc5 polypeptides, or fragments thereof, are fusedto an antibody (e.g., IgG 1, IgG2, IgG3, IgG4) fragment (e.g.,polypeptides). Techniques for preparing these fusion proteins are known,and are described, for example, in WO 99/31241 and in Cosman et al.(2001) Immunity 14:123-133. Fusion to an Fc polypeptide offers theadditional advantage of facilitating purification by affinitychromatography over Protein A or Protein G columns.

In still another embodiment, an Fndc5 polypeptide may be labeled with afluorescent label to facilitate their detection, purification, orstructural characterization. In an exemplary embodiment, an Fndc5polypeptide of the invention may be fused to a heterologous polypeptidesequence which produces a detectable fluorescent signal, including, forexample, green fluorescent protein (GFP), enhanced green fluorescentprotein (EGFP), Renilla Reniformis green fluorescent protein, GFPmut2,GFPuv4, enhanced yellow fluorescent protein (EYFP), enhanced cyanfluorescent protein (ECFP), enhanced blue fluorescent protein (EBFP),citrine and red fluorescent protein from discosoma (dsRED).

Another aspect of the invention pertains to the use of isolated Fndc5proteins, and biologically active portions thereof, as well as peptidefragments suitable for use as immunogens to raise anti-Fndc5 antibodies.An “isolated” or “purified” protein or biologically active portionthereof is substantially free of cellular material when produced byrecombinant DNA techniques, or chemical precursors or other chemicalswhen chemically synthesized. The language “substantially free ofcellular material” includes preparations of Fndc5 protein in which theprotein is separated from cellular components of the cells in which itis naturally or recombinantly produced. In one embodiment, the language“substantially free of cellular material” includes preparations of Fndc5protein having less than about 30% (by dry weight) of non-Fndc5 protein(also referred to herein as a “contaminating protein”), more preferablyless than about 20% of non-Fndc5 protein, still more preferably lessthan about 10% of non-Fndc5 protein, and most preferably less than about5% non-Fndc5 protein. When the Fndc5 protein or biologically activeportion thereof is recombinantly produced, it is also preferablysubstantially free of culture medium, culture medium represents lessthan about 20%, more preferably less than about 10%, and most preferablyless than about 5% of the volume of the protein preparation. Thelanguage “substantially free of chemical precursors or other chemicals”includes preparations of Fndc5 protein in which the protein is separatedfrom chemical precursors or other chemicals which are involved in thesynthesis of the protein. In one embodiment, the language “substantiallyfree of chemical precursors or other chemicals” includes preparations ofFndc5 protein having less than about 30% (by dry weight) of chemicalprecursors of non-Fndc5 chemicals, more preferably less than about 20%chemical precursors of non-Fndc5 chemicals, still more preferably lessthan about 10% chemical precursors of non-Fndc5 chemicals, and mostpreferably less than about 5% chemical precursors of non-Fndc5chemicals. In preferred embodiments, isolated proteins or biologicallyactive portions thereof lack contaminating proteins from the same animalfrom which the Fndc5 protein is derived. Typically, such proteins areproduced by recombinant expression of for example, a human Fndc5 proteinin a nonhuman cell.

In preferred embodiments, the protein or portion thereof comprises anamino acid sequence which is sufficiently homologous to an amino acidsequence of SEQ ID NO: 2, 4, 6, 8, 10, 12 or 14, or fragment thereof,such that the protein or portion thereof maintains one or more of thefollowing biological activities or, in complex, modulates (e.g.,enhance) one or more of the following biological activities: 1)BDNFexpression in the central and/or peripheral nervous system; 2)activity-induced immediate-early gene expression in neurons; 3) neuronalsurvival; 4) neurological lesion formation; 5) neurite outgrowth; 6)synaptogenesis; 7) synaptic plasticity; 8) neuronal mitochondrialfunction; 9) dendritic arborization; 10) neuronal differentiation; and11) neuronal migration. The portion of the protein is preferably abiologically active portion as described herein. In another preferredembodiment, the Fndc5 protein has an amino acid sequence shown in SEQ IDNO: 2, 4, 6, 8, 10, 12 or 14, or fragment thereof, respectively, or anamino acid sequence which is at least about 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or morehomologous to the amino acid sequence shown in SEQ ID NO: 2, 4, 6, 8,10, 12 or 14, or fragment thereof. In yet another preferred embodiment,the Fndc5 protein has an amino acid sequence which is encoded by anucleotide sequence which hybridizes, e.g., hybridizes: under stringentconditions, to the nucleotide sequence of SEQ ID NO:1, 3, 5, 7, 9, 11,13 or 15, or fragment thereof, or a nucleotide sequence which is atleast about 50%, preferably at least about 60%, more preferably at leastabout 70%, yet more preferably at least about 80%, still mom preferablyat least about 90%, and most preferably at least about 95% or morehomologous to the nucleotide sequence shown in SEQ ID NO: 1, 3, 5, 7, 9,11, 13 or 15, or fragment thereof. The preferred Fndc5 proteins of thepresent invention also preferably possess at least one of the Fndc5biological activities, or activities associated with the complex,described herein. For example, a preferred Fndc5 protein of the presentinvention includes an amino acid sequence encoded by a nucleotidesequence which hybridizes, e.g., hybridizes under stringent conditions,to the nucleotide sequence of SEQ ID NO:1, 3, 5, 7, 9, 11, 13 or 15, orfragment thereof and which can maintain one or more of the followingbiological activities or, in complex, modulates (e.g., enhance) one ormore of the following biological activities: 1) BDNF expression in thecentral and/or peripheral nervous system; 2) activity-induced immediateearly gene expression in neurons; 3) neuronal survival; 4) neurologicallesion formation; 5) neurite outgrowth; 6) synaptogenesis; 7) synapticplasticity; 8) neuronal mitochondrial function; 9) dendriticarborization; 10) neuronal differentiation; and 11) neuronal migration.

Biologically active portions of the Fndc5 protein include peptidescomprising amino acid sequences derived from the amino acid sequence ofthe Fndc5 protein, e.g., the amino acid sequence shown in SEQ ID NO: 2,4, 6, 8, 10, 12 or 14, or fragment thereof, or the amino acid sequenceof a protein homologous to the Fndc5 protein, which include fewer aminoacids than the full length Fndc5 protein or the full length proteinwhich is homologous to the Fndc5 protein, and exhibit at least oneactivity of the Fndc5 protein, or complex thereof. Typically,biologically active portions (peptides, e.g., peptides which are, forexample, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acidsin length) comprise a domain or motif, e.g., signal peptide,extracellular domain, fibronectin domain, hydrophobic, and/or C-terminaldomain). In a preferred embodiment, the biologically active portion ofthe protein which includes one or more the domains/motifs describedherein can increase 1) BDNF expression in the central and/or peripheralnervous system; 2) activity-induced immediate-early gene expression inneurons; 3) neuronal survival; 4) neurological lesion formation; 5)neurite outgrowth; 6) synaptogenesis; 7) synaptic plasticity; 8)neuronal mitochondrial function; 9) dendritic arborization; 10) neuronaldifferentiation; and 11) neuronal migration. Moreover, otherbiologically active portions, in which other regions of the protein aredeleted, can be prepared by recombinant techniques and evaluated for oneor more of the activities described herein. Preferably, the biologicallyactive portions of the Fndc5 protein include one or more selecteddomains/motifs or portions thereof having biological activity. In anexemplary embodiment, an Fndc5 fragment comprises and/or consists ofabout amino acids 29-140, 29-150, 30-140, 30-150, 73-140, 73-150, 1-140,1-150, or any range in between residues 1 and 150 of SEQ ID NO:2. Inanother embodiment, an Fndc5 fragment consists of a portion of afull-length Fndc5 fragment of interest that is less than 195, 190, 185,180, 175, 170, 165, 160, 155, 150, 145, 140, 135, 130, 125, 120, 115,110, 105, 100, 95, 90, 85, 80, 75, or 70 amino acids in length.

Fndc5 proteins can be produced by recombinant DNA techniques. Forexample, a nucleic acid molecule encoding the protein is cloned into anexpression vector (as described above), the expression vector isintroduced into a host cell (as described above) and the Fndc5 proteinis expressed in the host cell. The Fade5 protein can then be isolatedfrom the cells by an appropriate purification scheme using standardprotein purification techniques. Alternative to recombinant expression,an Fndc5 protein, polypeptide, or peptide can be synthesized chemicallyusing standard peptide synthesis techniques. Moreover, native Fndc5protein can be isolated from body fluids like plasma or cells (e.g.,neurons), for example using an anti-Fndc5 antibody (described furtherbelow).

Also provided are Fndc5 chimeric or fusion proteins. As used herein, anFndc5 “chimeric protein” or “fusion protein” comprises an Fndc5polypeptide operatively linked to a non-Fndc5 polypeptide. A “Fndc5polypeptide” refers to a polypeptide having an amino acid sequencecorresponding to Fndc5, whereas a “non-Fndc5 polypeptide” refers o apolypeptide having an amino acid sequence corresponding to a proteinwhich is not substantially homologous to the Fndc5 protein,respectively, e.g., a protein which is different from the Fndc5 proteinand which is derived from the same or a different organism. Within thefusion protein, the term “operatively linked” is intended to indicatethat the Fndc5 polypeptide and the non-Fndc5 polypeptide are fusedin-frame to each other. The non-Fndc5 polypeptide can be fused to theN-terminus or C-terminus of the Fndc5 polypeptide, respectively. Forexample, in one embodiment the fusion protein is a Fndc5-GST and/orFndc5-Fc fusion protein in which the Fndc5 sequences, respectively, arefused to the N-terminus of the GST or Fc sequences. Such fusion proteinscan facilitate the purification, expression, and/or bioavailability ofrecombinant Fndc5. In another embodiment, the fusion protein is an Fndc5protein containing a heterologous signal sequence at its C-terminus. Incertain host cells (e.g., mammalian host cells), expression and/orsecretion of Fndc5 can be increased through use of a heterologous signalsequence.

Preferably, an Fndc5 chimeric or fusion protein of the invention isproduced by standard recombinant DNA techniques. For example, DNAfragments coding for the different polypeptide sequences are ligatedtogether in-frame in accordance with conventional techniques, forexample by employing blunt-ended or stagger-ended termini for ligation,restriction enzyme digestion to provide for appropriate termini,filling-in of cohesive ends as appropriate, alkaline phosphatasetreatment to avoid undesirable joining, and enzymatic ligation. Inanother embodiment, the fusion gene can be synthesized by conventionaltechniques including automated DNA synthesizers. Alternatively, PCRamplification of gene fragments can be carried out using anchor primerswhich give rise to complementary overhangs between two consecutive genefragments which can subsequently be annealed and reamplified to generatea chimeric gene sequence (see, for example, Current Protocols inMolecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST polypeptide). AnFndc5-encoding nucleic acid can be cloned into such an expression vectorsuch that the fusion moiety is linked in-frame to the Fndc5 protein.

Also provided are homologues of the Fndc5 proteins which function aseither an Fndc5 agonist (mimetic) or an Fndc5 antagonist. In a preferredembodiment, the Fndc5 agonists and antagonists stimulate or inhibit,respectively, a subset of the biological activities of the naturallyoccurring form of the Fndc5 protein. Thus, specific biological effectscan be elicited by treatment with a homologue of limited function. Inone embodiment, treatment of a subject with a homologue having a subsetof the biological activities of the naturally occurring form of theprotein has fewer side effects in a subject relative to treatment withthe naturally occurring form of the Fndc5 protein.

Homologues of the Fndc5 protein can be generated by mutagenesis, e.g.,discrete point mutation or truncation of the Fndc5 protein. As usedherein, the term “homologue” refers to at variant form of the Fndc5protein which acts as an agonist or antagonist of the activity of theFndc5 protein. An agonist of the Fndc5 protein can retain substantiallythe same, or a subset, of the biological activities of the Fndc5protein. An antagonist of the Fndc5 protein can inhibit one or more ofthe activities of the naturally occurring form of the Fndc5 protein, by,for example, competitively binding, to a downstream or upstream memberof the Fndc5 cascade which includes the Fndc5 protein. Thus, themammalian Fndc5 protein and homologues thereof of the present inventioncan be, for example, either positive or negative regulators of adipocytedifferentiation and/or thermogenesis in brown adipocytes.

In an alternative embodiment, homologues of the Fndc5 protein can beidentified by screening combinatorial libraries of mutants, e.g.,truncation mutants, of the Fndc5 protein for Fndc5 protein agonist orantagonist activity. In one embodiment, a variegated library of Fndc5variants is generated in combinatorial mutagenesis at the nucleic acidlevel and is encoded by a variegated gene library. A variegated libraryof Fndc5 variants can be produced by, for example, enzymaticallyligating a mixture of synthetic oligonucleotides into gene sequencessuch that a degenerate set of potential Fndc5 sequences is expressibleas individual polypeptides, or alternatively, as a set of larger fusionproteins (e.g., for phage display) containing the set of Fndc5 sequencestherein. There are a variety of methods which can be used to producelibraries of potential Fndc5 homologues from a degenerateoligonucleotide sequence. Chemical synthesis of a degenerate genesequence can be performed in an automatic DNA synthesizer, and thesynthetic gene then ligated into an appropriate expression vector. Useof a degenerate set of genes allows for the provision, in one mixture,of all of the sequences encoding the desired set of potential Fndc5sequences. Methods for synthesizing degenerate oligonucleotides areknown in the art (see, e.g., Narang, S. A, (1983) Tetrahedron 39:3;Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984)Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477.

In addition, libraries of fragments of the Fndc5 protein coding can beused to generate a variegated population of Fndc5 fragments forscreening and subsequent selection of homologues of an Fndc5 protein. Inone embodiment, a library of coding sequence fragments can be generatedby treating a double stranded PCR fragment of an Fndc5 coding sequencewith a nuclease under conditions wherein nicking occurs only about onceper molecule, denaturing the double stranded DNA, renaturing the DNA toform double stranded DNA which can include sense/antisense pairs fromdifferent nicked products, removing single stranded portions fromreformed duplexes by treatment with SI nuclease, and ligating theresulting fragment library into an expression vector. By this method, anexpression library can be derived which encodes N-terminal, C-terminaland internal fragments of various sizes of the Fndc5 protein.

Several techniques are known in the art for screening gene products ofcombinatorial libraries made by point mutations or truncation, and forscreening cDNA libraries for gene products having a selected property.Such techniques are adaptable for rapid screening of the gene librariesgenerated by the combinatorial mutagenesis of Fndc5 homologues. The mostwidely used techniques, which are amenable to high through-put analysis,for screening large gene libraries typically include cloning the genelibrary into replicable expression vectors, transforming appropriatecells with the resulting library of vectors, and expressing thecombinatorial genes under conditions in which detection of a desiredactivity facilitates isolation of the vector encoding the gene whoseproduct was detected. Recursive ensemble mutagenesis (REM), a newtechnique which enhances the frequency of functional mutants in thelibraries, can be used in combination with the screening assays toidentify Fndc5 homologues (Arkin and Youvan (1992) Proc. Natl. Acad.Sci. USA 89:7811-7815; Delagrave et al. (1993) Protein Engineering6(3):327-331).

In addition, useful host cells and vectors are described supra forexpressing desired nucleic acids and proteins for use according to themethods described herein.

Exemplification

This invention is further illustrated by the following examples, whichshould not be construed as limiting.

EXAMPLE 1 Materials and Methods for Examples 1-10 A. Reagents

All primers used are listed with their sequences in Table 2. Recombinanthuman BDNF was purchased from PeproTech. Recombinant human GDNF and CNTFand forskolin were obtained from Sigma. Recombinant mouse IGF-1 wasobtained, from R&D Systems. Recombinant mouse NGF and K252a wereobtained from EMD Nifedipine, XCT 790, DY 131, GW7647, and GW0742 werepurchased from Tocris. Recombinant irisin (human, rat, mouse, canine)was obtained from Phoenix Pharmaceuticals (Burlingame, Calif.).

TABLE 2 Primer Sequence (5′ to 3′) mRps18 QS CATGCAGAACCCACGACAGTAmRps18 AS CCTCACGCAGCTTGTTGTCTA mFndc5QS ATGAAGGAGATGGGGAGGAA mFndc5QAGCGGCAGAAGAGAGCTATAACA mPGC-1aQS TGATGTGAATGACTTGGATACAGACA mPGC-1aQAGCTCATTGTTGTACTGGTTGGATATG mErra QS CACTACGGTGTGGCATCCTG mErra ASACAGCTGTACTCGATGCTCC mErrb QS AACCGAATGTCGTCCGAAGAC mErrb ASGTGGCTGAGGGCATCAATG mErrg QS ATGGATTCGGTAGAACTTTGCC mErrg ASCTTCTTCGTAGTGCAGGGAAAA mBdnf QS TGGCCCTGCGGAGGCTAAGT mBdnf ASAGGGTGCTTCCGAGCCTTCCT mIgf1 QS TGGATGCTCTTCAGTTCGTG mIgf1 ASGTCTTGGGCATGTCAGTGTG mNpas4QS CTGCATCTACACTCGCAAGG mNpas4QAGCCACAATGTCTTCAAGCTCT mc-FosQS ATGGGCTCTCCTGTCAACACAC mc-FosQAATGGCTGTCACCGTGGGGATAAAG mArcQS TACCGTTAGCCCCTATGCCATC mArcQATGATATTGCTGAGCCTCAACTG mZif268QS TATGAGCACCTGACCACAGAGTCC mZif268QACGAGTCGTTTGGCTGGGATAAC Key QS: qPCR-sense QA: qPCR-antisense

In addition to the Fndc5 sequences described herein (e.g., Table 1), thefollowing sequences are useful for generating the constructs used in theexperiments described below.

For example, the IrisinFc constructs encode or are composed of thefollowing amino acid sequence:

DSPSAPVNVTVRHLKANSAVVSWDVLEDEVVIGFAISQQKKDVRMLRFIQEVNTTTRSCALWDLEEDTEYIVHVQAISIQGQSPASEPVLFKTPREAEKMASKNKDEVTMKEGGGGAGGGGVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

The control human Fc (hFc) constructs encode or are composed of thefollowing amino acid sequence:

VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

The GFP sequence used in the GFP adenovirus constructs are encoded bythe following nucleic acid sequence:

AAGCTTGGGATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAACGCGGATCCACTAGTTCTAGAGC.

Additional information as to construct generation and sequences, such asFc fusion constructs, are described in Bostrom et al. (2012) Nature 481,463-468.

B. Animal Studies

All animal experiments were performed according to procedures approvedby the IACUC of Dana-Farber Cancer Institute and the BIDMC. Generationand characterization of the Pgc1a total body KO (Pgc1a^(−/−)) mice havebeen described previously in Lin et al. (2004) Cell 119, 121-135. Micewere kept under 14-hour light/10-hour dark cycles at constanttemperature (22° C.) with free access to food and water. Mice were fed astandard diet (Rodent Diet 8664, Harlan Teklad). For free wheel runningexercise, six week old male wild type C56/B16 mice (Jackson Laboratory)were housed individually with stainless steel running wheels. Sedentarycontrols were housed without wheels. Mice were exercised for 30 days andsacrificed approximately 10 h after their last bout of exercise and theindicated tissues were harvested. For the tissue panel, 13 weeks oldmale C57/B16 mice were used. For the developmental time-course pups weresacrificed at the indicated time-points and brains were harvested fortotal RNA. For RNA expression studies, animals were sacrificed andtissues harvested and stored at −80° C. until analysis.

C. Cell Culture

Primary cortical and hippocampal neurons were isolated as described ingreat detail previously in Bartlett and Banker (1984) J. Neurosci. 4,1944-1953. Briefly, cortices and hippocampi were dissected from E16-E18embryos, dissociated with trypsin (Sigma) and DNAse (Roche), and platedon poly-L-lysine-coated (Sigma) plates. Dissociated neurons werecultured in Neurobasal Media supplemented with B27, GlutaMAX™ (LifeTechnologies), and Penicillin-Streptomycin (Cellgro).

D. RNA Preparation and Expression Analysis

Cells or tissues were lysed and homogenized in TRIzol (Invitrogen).Total RNA was subsequently isolated using the RNeasy Mini or Micro Kit(Qiagen). First-strand cDNA was generated using the High Capacity cDNAReverse Transcription Kit (Life Technologies), and qPCR was performedusing SYBR Green Master Mix in a 7900HT Real-Time PCR system (AppliedBiosystems). mRNA quantities were normalized to Rsp18 afterdetermination by the comparative Ct method (Schmittgen and Livak (2008)Nat. Protocols 3, 1101-1108).

E. Protein Extraction and Western Blot Analysis

Cell lysates were prepared with RIPA buffer supplemented with completeprotease inhibitor cocktails. For generation of conditioned media cellswere washed three times with PBS and plain neurobasal with glutamine andantibiotics but without B27 supplement was added. Cells and media werecollected the next morning. The conditioned media was spun twice at lowspeed and then concentrated in spin-filter columns with a molecularweight cut-off of 3 KDa Deglycosylation was performed using ProteinDeglycosylation Mix (New England Biolabs). Blood was collected inlithium heparinized tubes (BD Biosciences) and plasma was separated bycentrifugation. Albumin and IgG was removed using the ProteoExtract-kit(Millipore). Then the samples were concentrated using Ultra-2Centrifugal Filter (Millipore) and deglycosylated with PNGase F (NewEngland Biolabs).

For Western blot analyses, 80-100 pg protein was subjected to SDS-PAGEunder reducing conditions, transferred, and blotted with anti-PGC-1αmouse (4C1.3) antibody (Calbiochem/EMD Millipore,) and anti-FNDC5(Irisin) rabbit polyclonal antibody (Adipogen). Equal loading wasassessed by Ponceau staining (Sigma-Aldrich).

F. Forced Expression and Knockdown

Generation and delivery of the PGC-1α, GFP, and FNDC5 adenovirus hasbeen described in detail in Bostrom et al. (2012) Nature 481, 463-468and Lustig et al. (2011) Genes Dev. 25, 1232-1244.

Primary cortical neurons were transduced at the indicated time-pointsand were harvested 48 hrs later for RNA isolation. For knockdownstudies, primary cortical neurons were transduced with viralsupernatants from HEK293T cells transfected with pLKO.1 vector (TRC)containing the specified shRNAs at the indicated time-points. Thesequences of shRNAs used are as follows:

Forward Reverse Target Oligo  Oligo Sequence Sequence Sequence (5′to 3′) (5′ to 3′) (5′ to 3′) shFndc5-1 CCCTCTGTGA CCGGCCCTCT AATTCAAAAAACATCATCAA GTGAACATCA CCCTCTGTGA A TCAAACTCGA ACATCATCAA GTTTGATGATACTCGAGTTT GTTCACAGAG GATGATGTTC GGTTTTTG ACAGAGGG shFndc5-2 GTGCGGATGCCCGGGTGCGG AATTCAAAAA TCCGGTTCAT ATGCTCCGGT GTGCGGATGC T TCATTCTCGATCCGGTTCAT GAATGAACCG TCTCGAGAAT GAGCATCCGC GAACCGGAGC ACTTTTTG ATCCGCACshFndc5-3 CGAGCCCAAT CCGGCGAGCC AATTCAAAAA AACAACAAGG CAATAACAACCGAGCCCAAT A AAGGACTCGA AACAACAAGG GTCCTTGTTG ACTCGAGTCC TTATTGGGCTTTGTTGTTAT CGTTTTTG TGGGCTCG

Cells were harvested four days later for total RNA. To producelentiviral supernatants, HEK293T cells cultured in DMEM with 10% FBS,were transfected using Lipofectamine2000™ (Life Technologies) with thespecified shRNA plasmid and the packing plasmid psPAX2 and pMD2.G in a2:1:1 ratio. After an overnight incubation, media was exchanged toneurobasal media supplemented as described above and supernatants wereharvested 24 hrs later.

G. Cell Viability Assay

Cell viability of cultured neurons was assessed using CellTiter-Glo®Luminescent Cell Viability Assay (Promega, Madison, Wis.) according tothe manufacturer's instructions. Luminescence of cell lysates wasmeasured using the FLUOstar Omega plate reader (BMG LABTECH, Offenburg,Germany).

H. Analysis of the Murine Fndc5 Promoter for Erra Transcription FactorBinding Sites

The genomic sequence of the murine Fndc5 gene and 6 kb of its upstreampromoter was retrieved from the USCS Genome browser (available on theWorld Wide Web at genome.ucsc.edu; assembly mm9). This genomic sequencewas searched for the canonical Erra transcription facto binding motif:TGACCTT. This motif had been identified and established in previousstudies described in Charest-Marcotte et al. (2010) Genes Dev. 24,537-542; Mootha et al. (2004) Proc. Natl. Acad. Sci. 101, 6570-6575; andWang et al. (2012) Genome Res. 22, 1798-1812.

I. Peripheral Delivery of FNDC5 by Adenoviral Vectors

High titer GFP- or FNDC5-expressing adenoviral particles were obtainedby ViraQuest Inc. (North Liberty, Iowa). Five week old male wild-typeBALB/c mice were injected with GFP- or FNDC5-expressing adenoviralparticles (10¹¹/animal) intravenously. Animals were sacrificed sevendays later and the indicated tissues were harvested for gene expressionanalyses using qPCR.

J. Stem Cell Differentiation and Glial Co-Culture

Growth of human embryonic stem cells, differentiation of human embryonicstem cells into motor neurons, and glial co-culture were performed asdescribed in DiGiorgio et al. (2008) Cell Stem Cell 3:637-648.

EXAMPLE 2 Endurance Exercise Induces Hippocampal Fndc5 Gene Expression

Although FNDC5 is highly expressed in the brain, as well as in skeletalmuscle (Ferrer-Martinez et al. (2002) Dev. Dyn. 224, 154-167 and Teufelet al. (2002) Gene 297, 79-83), very little is known about its functionin the brain. In order to investigate the effects of exercise on FNDC5expression and function, an established endurance exercise regimen of 30days of voluntary free running-wheel exercise was used. This regimen isknown to induce BDNF expression, neurogenesis, dendritic spines andimproved memory function in mice (Eadie et al. (2005) J. Comp. Neurol.486, 39-47 and Kobilo et al. (2011) Learning Mem. (Cold Spring Harbor,N.Y.) 18, 605-609). As has previously been established, this trainingwas sufficient to induce muscle Fndc5 gene expression (FIG. 1A), as wellas the transcriptional regulators Pgc1a and Erra, known mediators of theexercise-response in skeletal muscle. In addition, other known genes ofthe exercise gene program were induced, confirming an adaptive enduranceexercise response in the muscle (FIG. 2). The same exercise regime ledto a significant elevation of Fndc5 expression in the hippocampus (FIG.1B) but not in the remainder of the brain (FIG. 1C). The hippocampus isa region of the brain involved in learning and memory and has beenidentified as a major site where changes induced by exercise occur. Eventhough genes that are induced by neuronal activity, such as Arc, cFosand Zif268, were upregulated in both the remainder of the brain and thehippocampus, the important exercise-related neurotrophin Bdnf wasinduced only in the hippocampus (FIGS. 1D-1E). However, Npas4, animportant transcriptional component in hippocampal function and a keyregulator of activity-induced Bdnf expression (Lin et al. (2008) Nature455, 1198-1204 and Ramamoorthi et al. (2011) Science 334, 1669-1675) wasnot increased in the exercise regimen used here (FIGS. 1D-1E). Thesedata indicate that the induction of FNDC5 is part of the transcriptionalresponse to exercise in the hippocampus.

EXAMPLE 3 Fndc5 Gene Expression Correlates with Pgc1a Expression Levelsin Various Tissues and Developmental Stages

It was previously reported that elevations in Fndc5 gene expression inexercised muscle was dependent on PGC-1α (Bostrom et al. (2012) Nature481, 463-468). It was therefore investigated whether Fndc5 expression inthe brain is also regulated by PGC-1α. To first assess if there is acorrelation between the gene expression of these two proteins, differenttissues were isolated from C57/B16 mice, total RNA was extracted, andgene expression was measured for Fndc5 and Pgc1a. Consistent withearlier reports, the highest level of Fndc5 gene expression was detectedin heart, skeletal muscle, brain and spinal cord (Ferrer-Martinez et al.(2002) Dev. Dyn. 224, 154-167 and Teufel et al. (2002) Gene 297, 79-83).When the different tissues were grouped according to their levels ofFndc5 expression, most tissues with very high Fndc5 expression alsoshowed relatively high levels of Pgc1a gene expression (FIG. 3A). Fndc5and Pgc1expression levels correlated well, even within very distinctmuscle beds. Fndc5 expression was higher in oxidative muscle, such asthe soleus muscle, which also contains higher levels of Pgc1a, than inglycolytic or mixed muscles, such as gastrocnemius or quadriceps muscle.Exceptions to this tight correlation of Fndc5 and Pgc1a expression arethe interscapular brown adipose tissue and the kidney. Both are tissueswith extremely high mitochondrial content, which might explain theirrequirement for high Pgc1a levels without very high expression of Fndc5.

To examine whether FNDC5 and PGC-1α were developmentally regulated insynchrony during maturation of the brain, a time-course experiment ofpostnatal development was performed. Brains were harvested from pups atpostnatal day 0 (P0), P10, P20, P25, and P30 and gene expression wasmeasured by qPCR. These time-points were chosen because they cover animportant time period of postnatal brain developmental, up to the maturestate at P30. A two-step pattern of increased Fndc5 gene expressionduring development was observed, with a first increase between P0 andP10 and second increase between P10 and P20, which then leveled off(FIG. 3B). Pgc1a gene expression followed essentially the same pattern.This two-step pattern of increased gene expression during braindevelopment was also observed for the key neural regulatory protein,Bdnf. Next, the gene expression patterns for these factors were assessedduring the maturation of primary cortical neurons in culture. Thecorrelation was observed again: Fndc5 gene expression increased betweenin vitro days (DIV) 1 and DIV 6, when the expression levels of Pgc1a andBdnf were also elevated (FIG. 3C). These data illustrate that, similarto muscle, there is a strong correlation between PGC-1α and FNDC5 geneexpression in the brain.

EXAMPLE 4 Neuronal Fndc5 Gene Expression is Regulated by PGC-1α

To investigate whether PGC-1α is a transcriptional regulator of Fndc5gene expression in the brain, dissociated primary conical neurons inculture were used. Although more heterogeneous than neurons from thedentate gyrus of the hippocampus, these cultures can be isolated insufficient quantities for molecular studies and can be readilymanipulated Primary cortical neurons were stimulated with forskolin (10μM), a strong inducer of intracellular cAMP, which is known to increasePgc1a gene expression in cell types as diverse as brown adipocytes,hepatocytes and Schwann cells (Cowell et al. (2008) Neurosci. Lett. 439,269-274; Herzig et al. (2001) Nature 413, 179-183: and Yoon et al.(2001) Nature 413, 131-138). This increase in Pgc1a gene expression wasaccompanied by a significant increase in Fndc5 gene expression (FIG.4A). On the other hand, treatment of cortical neurons with nifedipine (5μM), a selective L-type calcium channel blocker, which leads todecreased intracellular calcium levels and decreased Pgc1a geneexpression, was accompanied by decreased Fndc5 gene expression (FIG.4B).

Next, genetic gain- and loss-of-function approaches were used to testcausality. Forced expression of PGC-1α by adenoviral deliver in primarycortical neurons resulted in a 4-fold increase in Fndc5 gene expression(FIG. 4C). Immunoblotting confirmed that the increase in Fndc5 mRNAtranslated into elevated FNDC5 protein levels (FIG. 5). Conversely,reducing Pgc1a gene expression with lentiviral-mediated shRNA knockdownby more than 40% significantly decreased Fndc5 gene expression by 66 and31%, respectively (FIG. 4D). As an additional loss-of-function model,the brains of global Pgc1a knockout mice (Pgc1a−/−) were used. The samerequirement of PGC-1α for Fndc5 gene expression in brains of these mice,which display a reduction in Fndc5 gene expression by 32%, was observed(FIG. 4E). Taken together, these results demonstrate that PGC-1α is aregulator of neuronal Fndc5 gene expression in neural cultures and inthe brain.

EXAMPLE 5 ERRα is a Key Interacting Transcription Factor with PGC-1α forRegulating Fndc5 Gene Expression in Neurons

PGC-1α is a transcriptional co-activator, meaning it does not bind tothe DNA itself but interacts with transcription factors to execute itseffects on gene expression (Spiegelman (2007) Novartis FoundationSympos. 287, 60-69). The orphan nuclear receptor estrogen-relatedreceptor alpha (ERRα; also known as NR3B1) is a central metabolicregulator (Giguere et al. (1988) Nature 331, 91-94 and Luo et al. (2003)Mol. Cell. Biol. 23, 7947-7956) and a very important interactor withPGC-1α (Laganiere et al. (2004) J. Biol. Chem, 279, 18504-18510: Moothaet al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 6570-6575; andSchreiber et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 6472-6477).The interaction of Errα with PGC-1α has been best studied in skeletalmuscle, where it is required for mitochondrial biogenesis, induction ofangiogenesis, oxidative metabolism, and oxidative muscle fibers (Aranyet al. (2008) Nature 451, 1008-1012; Mootha et al. (2004) Proc. Natl.Acad. U.S.A. 101, 6570-6575; and Schreiber et al. (2004) Proc. Natl.Acad. Sci. U.S.A. 101, 6472-6477).

Erra follows the exercise-induced gene expression pattern of Fndc5 inthe brain. Erra is up-regulated in the hippocampus upon exercise but notin the rest of the brain (FIGS. 1B-1C). In addition, there was acorrelation between Fndc5 and Erra gene expression in the tissue-panel(FIG. 3A) as well as in the developmental time-course (FIG. 3B). PGC-1αis well-known to often increase the expression of transcription factorsthat it interacts with, thereby positively regulating its own regulators(Handschin et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 7111-7116and Mootha et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 6570-6575).It was therefore asked if forced expression of PGC-1α in primarycortical neurons results in an increase Erra mRNA. Indeed, adenoviralexpression of PGC-1α significantly increased Erra gene expression, butnot Errb or Errg gene expression (FIG. 6A). However, mRNA for othercommon binding partners of PGC-1α, such as Mef2, Ppara, Nrf1 or Gabpa/bwas not induced in these experiments (FIG. 7A).

The murine Fndc5 gene and 6 kb of its upstream promoter were searchedfor putative ERRα transcription factor binding sites, (ERRE), with thecanonical ‘TGACCTT’ sequence (Charest-Marcotte et al. (2010) Genes Dev.24, 537-542; Mootha et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101,6570-6575; and Wang et al. (2012) Genome Res. 22, 1798-1812). Twoputative ERRE's were identified: one around 5.3 kb upstream of thetranscriptional start site and one in the fourth intron of the Fndc5gene (FIG. 6B). ERRα had been previously reported to also bind tointronic sequences to exert its biological function (Arany et al. (2008)Nature 451, 1008-1012). This further indicates that ERRα is important inFNDC5 gene regulation.

Treatment of primary cortical neurons with XCT 790 (1 μM), a selectiveERRα inhibitor (inverse agonist), which disrupts the ERRα/PGC-1αtranscriptional complex (Mootha et al. (2004) Proc. Natl. Acad. Sci.U.S.A. 101, 6570-6575), significantly reduced Fndc5 gene expressioncompared to vehicle treated cells (FIG. 6C). However, stimulation withDY131 (1 μM), a selective ERRβ and ERRγ agonist, had no effect on Fndc5gene expression. This results indicates certain specificity for theinvolvement of ERRα compared to other ERR subfamily members. Since thenuclear receptor PPARα, another common binding partner of PGC-1α, wasslightly induced by forced expression of PGC-1α, the effect of GW7647, apotent and highly selective PPARα agonist, and GW0742, a potent andhighly selective PPARδ agonist were tested on Fndc5 gene expression.However, under the conditions tested, no effect on Fndc5 gene expressionin primary cortical neurons by these compounds was observed (FIG. 7B).

The results from the treatment with ERRα antagonist indicate thatinteraction of the PGC-1α with ERRα is required for the PGC-1α-dependentinduction of Fndc5 gene expression To test this, ERRα was first knockeddown in primary cortical neurons using lentivirally expressed shRNAhairpins and then three days later the cells were transduced with eitherthe PGC-1α adenovirus or GFP expressing adenovirus. Erra mRNA wasefficiently knocked-down by this hairpin (70%) and forced expression ofPGC-1α did not affect the efficiency of the knock-down (FIG. 3CC).Knockdown of ERRα significantly reduced Fndc5 gene expression at baseline (FIG. 6D). Furthermore, forced expression of PGC-1α by adenovirusin the cells with reduced ERRα failed to significantly increase Fndc5gene expression (FIG. 6D). However, this failure to increase Fndc5 geneexpression was not due to a lack over expression of PGC-1α in the shErratreated neurons (FIG. 7C).

EXAMPLE 6 FNDC5 Regulates Bdnf Gene Expression in a Cell-AutonomousManner and Recombinant BDNF Decreases Fndc5 Gene Expression as Part ofPotential Feedback Loop

As described above, BDNF is a major mediator of certain beneficialeffects on the brain. In addition, an increase in the Bdnf geneexpression in the hippocampus was observed, where Fndc5 gene expressionwas also induced (FIGS. 1B-1D), but not in the rest of the brain, whereFndc5 was not induced (FIGS. 1C-1E). It was therefore tested whetherFNDC5 could be a regulator of Bdnf gene expression in a cell culturemodel. Primary cortical neurons were transduced with either FNDC5adenovirus or a GFP adenovirus as control. Forced expression of FNDC5resulted in a clear increase in FNDC5 protein in the whole cell lysate,as well as an increase in the secreted form of FNDC5 (irisin) in thecell culture supernatant (FIG. 8A). After deglycosylation, this proteinhad the same apparent molecular mass (2 kDa) as predicted for irisin(FIG. 8A). In addition, forced expression of FNDC5 significantlyupregulated Bdnf gene expression by four fold (FIG. 8B). Importantly,FNDC5 expression also induced other important activity-induced genesinvolved in hippocampal function including Npas4, cFos, and Arc.However, Zif268 was only slightly elevated.

Primary cortical neurons treated with 5× concentrated conditioned mediafrom CHO cell lines overexpressing either irisinFc or human Fc (hFc) asa control showed increased expression of Fndc5 and Bdnf (FIG. 13). Inaddition, FIG. 14 demonstrates that neurons bind irisinFc. Moreover,primary cortical neurons treated with either irisinFc during in vitroculture for 7 days showed significantly increased cell viabilityrelative to treatment with hFc (FIG. 15).

To investigate if FNDC5 is required for Bdnf gene expression,lentivirally delivered shRNA was used to knockdown FNDC5 in primarycortical neurons. To address possible off-targets of a single hairpin, atotal of five hairpins of which three significantly knocked down Fndc5mRNA (FIG. 8C). The same three hairpins also significantly reduced Bdnfgene expression. The role of PGC-1α in controlling Bdnf gene expressionin vivo was also analyzed. To do this, the brains of global Pgc1aknockout mice (Pgc1a^(−/−)) were used. As shown in FIG. 4E, Bdnf geneexpression was significantly reduced in the brains of Pgc1a^(−/−) mice.

BDNF is well-known for its ability to improve survival of neurons inculture. Thus, the effects of gain- and loss-of-function of FNDC5 oncell viability of cultured neurons were assessed using aluminescence/ATP-based assay. Gain-of-function of FNDC5 significantlyimproved neuron survival in culture (FIG. 8D), while loss-of-function ofFNDC5 using shRNA mediated knockdown of FNDC5 with two differenthairpins significantly impaired the survival of neurons in culture (FIG.8E).

To examine how BDNF might, in turn, alter FNDC5 gene expression, primarycortical neurons were stimulated with recombinant BDNF overnight atvarious concentrations at physiological and pharmacological dosages(0.1-100 ng/ml). BDNF concentrations as low as 1 ng/ml significantlyreduced Fndc5 gene expression (FIG. 8F) and a dose-response wasobserved. To ask whether the reduction in Fndc5 gene expression wasspecific to BDNF, primary cortical neurons were treated with a varietyof central and peripheral neurotrophic factors in addition to BDNF, suchas CNTF (ciliary neurotrophic factor), GDNF (glial cell-derivedneurotrophic factor), NGF (nerve growth factor), and IGF-1 (insulin-likegrowth factor 1) at 100 ng/ml for overnight. However, only BDNFstimulation significantly reduced Fndc5 mRNA expression (FIG. 8G). Thiseffect was abolished by pre-incubating the cortical neurons with a lowdose (50 nM) of K252a, well-characterized inhibitor of TrkB, thereceptor of BDNF signaling (Gimenez-Cassina et al. (2012) Neurosci.Lett. 531, 182-187 and Tapley et al. (1992) Oncogene 7, 371-381) (FIG.8H). In summary, these data indicate a homeostatic FNDC5/BDNF feed-backloop.

EXAMPLE 7 Peripheral Delivery of FNDC5 by Adenoviral Vectors IncreasesBdnf Expression in the Central Nervous System, Including theHippocampus, Cerebellum, and Sciatic Nerve

It was previously shown that adenoviral overexpression of FNDC5 in theliver, a major secretory organ, increases circulating levels of irisin,the secreted form of FNDC5 (Bostrom et al. (2012) Nature 481, 463-468).This resulted in the activation of a thermogenic gene program in certainfat tissues. To determine if peripheral delivery of FNDC5/irisin couldelevate central BDNF levels, adenoviral overexpression of FNDC5 in theliver was conducted and Bdnf gene expression in the hippocampus wasmeasured seven days later. As previously shown, forced expression ofFNDC5 in the liver resulted in the induced ‘browning’ of the inguinalfat depot (FIG. 9A), including increased expression of mRNA for a groupof key thermogenic genes, such as Pgc1a, Ucp1 and Cidia. In addition,plasma levels of irisin were elevated in mice overexpressing FNDC5 ascompared to GFP-overexpressing control mice (FIG. 10A).

Interestingly, Bdnf expression in the hippocampus was significantlyincreased, as was expression of Npas4, cFos, Are, and Zif268, all partof the activity-induced immediate early gene (IEG) program as mentionedbefore. Importantly, this was not caused by any viral-mediatedexpression of Fndc5 in the brain or hippocampus (FIG. 9B), indicatingthat the secreted form of the peripherally-expressed FNDC5 wasresponsible for the observed effect. This effect of increased Bdnfexpression was specific to the hippocampus and was not observed in theforebrain (FIG. 9C), whereas the IEG response was observed in both,which is consistent with the findings of the exercise effects describedabove (FIGS. 1D-1E). Similarly, peripheral injections of irisinFc causeda significant increase in Bdnf expression in the cerebellum (FIG. 16)and sciatic nerve (FIG. 17).

EXAMPLE 8 PGC-1α/FNDC5/BDNF Pathway in Primary Hippocampal Neurons

Cortical neurons were used in the experiments described above becausethis is the most widely used system of primary CNS cultures and becausereasonable numbers of cells can be obtained. However, since some of thedescribed in vivo observations were made in the hippocampus, thefindings were validated in primary hippocampal neurons.

Therefore, a key set of experiments were repeated in primary hippocampalneuron cultures. It was confirmed that Fndc5 gene expression issignificantly increased in primary hippocampal neurons cultured in vitrofrom DIV 1 to DIV6 and that the expression of Pgc1a and Bdnf mRNA issimilarly increased (FIG. 11A). To test whether PGC-1α regulates Fndc5gene expression in hippocampal neurons, gain- and loss of functionstudies were performed. Forced expression of PGC-1α significantlyinduced Fndc5 gene expression (FIG. 11B). Stimulation with forskolin (10μM) failed to induce Pgc1a gene expression, but decreased the expressionof Erra and Fndc5 (FIG. 12). Efficient knockdown of Pgc1a bylentivirally-delivered shRNA significantly reduced Fndc-5 geneexpression (FIG. 11C). Stimulation of primary hippocampal neurons withcommercially available recombinant irisin induced a similar gene program(Arc, cFos, Npas4, and Zif268) as was found in the in vivo adenoviralexperiments (FIG. 11D). However, the increase in Bdnf gene expressiondid not reach statistical significance. Loss-of-function of FNDC5 byshRNA mediated-knockdown with three different hairpins against Fndc5significantly reduced Bdnf gene expression in hippocampal neurons (FIG.11E). In addition, treatment of hippocampal neurons with recombinantBDNF reduced Fndc5 gene expression (FIG. 11F). Together, these datademonstrate that the basic observations made in the primary corticalneurons also apply to primary hippocampal neuron cultures.

EXAMPLE 9 Fndc5 is Functionally Associated with NeurodegenerativeDisorders

In addition to promoting neuronal survival described above, Fndc5expression and activity modulates neurons in neurodegenerativedisorders. MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) is aneurotoxin that destroys dopaminergic neurons in the substantia nigra ofthe brain to thereby model Parkinson's disease (St-Pierre et al. (2006)Cell 127, 397-408). FIG. 18 shows that mice treated with MPTP havesignificantly lower Fndc5 expression in their substantia nigra comparedto control mice not treated with MPTP in a human dopaminergic neuronalcell line. SH-SSY5Y, commonly used as a research model of Parkinson'sdisease, Fndc5 gene expression increases during differentiation ofSH-SSY5Y neurons with retinoic acid (FIG. 19) and treatment of SH-SSY5Yneurons with the neurotoxin rotenone commonly used to induceexperimental Parkinsonism in animals reduces Fndc5 gene expression (FIG.20; Krueger et al. (1990) Biochem. Biophys. Res. Comm. 169:123-128; andSamantaray et al. (2007) Neurosci. 146:741-755).

Fndc5 also has effects on motor neuron differentiation and synapseformation. For example, human embryonic stein cells differentiated intomotor neurons (eMN) show Fndc5 gene expression in response to irisin(FIG. 21) and, in response, such irisin activity on eMN promotes motorneuron differentiation and increases synapse formation (FIG. 22).

A recent study has reported a positive correlation between human brainsize and endurance exercise capacity suggesting a co-evolution betweenhuman cognition and locomotion (Raichlen and Gordon (2011) PLoS ONE 6,e20601). More complex tasks require a more complex brain and foraging inwide and open spaces in the savannas put high demands on spatialorientation, as well as the ability to acquire and retain newinformation. Therefore individuals with a more complex brain whoperformed better at these tasked might have had an evolutionaryadvantage. On the other hand, since endurance exercise clearly increasesexpression of BDNF in the brain, improvements in the exercise capacitymight have positively enforced brain growth (Mattson (2012) Aging Res.Rev. 11, 347-352), especially in the hippocampus.

A PGC-1α/FNDC5/BDNF pathway is described herein that is activated in thehippocampus by endurance exercise (FIG. 9). In this model, exerciseleads to increased transcription of Pgc1a and Erra. It has been observedpreviously that PGC-1α often induces the expression of transcriptionfactors to which it binds and co-activates (Handschin et al. (2003)Proc. Natl. Acad. Sci. U.S.A. 100, 7111-7116 and Mootha et al. (2004)Proc. Natl. Acad. Sci. U.S.A. 101. Indeed, the ability of PGC-1α toinduce FNDC5 gene expression depends on ERRα availability (FIG. 6D).This PGC-1α/Erra complex, in turn, likely binds to one or more of thecanonical ERRE's found in or near the Fndc5 gene, thus activating Fndc5gene expression. As shown in a cell culture model in FIG. 8A, FNDC5 is apositive regulator of BDNF expression. Based on this, it is believedthat the increased Fndc5 gene expression in exercise will lead toincreased BDNF levels. BDNF also can signal to reduce the expression ofFNDC5 as part of an apparent homeostatic loop. However, it is believedthat there are both FNDC5-dependent and FNDC5-independent pathways bywhich exercise induces BDNF expression. For example, CREB and NF-kB aretwo other transcription factors known to induce BDNF expression inexercise (Mattson (2012) Aging Res. Rev. 11, 347-352). These may actupstream or downstream of FNDC5, or in an independent pathway.

The induction of FNDC5 by exercise in the hippocampus is quantitativelycomparable to the induction observed in skeletal muscle It is also inthe same quantitative range as the induction of BDNF, a neurotrophicmediator of exercise in the brain, as well as cFos, Arc, and Zif268,important indicators for the activity state of neurons (Hunt et al.(1987) Nature 328, 632-634; Lyford et al. (1995) Neuron 14, 433-445;Rusak et al. (1990) Science 248, 1237-1240; and Saffen et al. (1988)Proc. Natl. Acad. Sci. U.S.A. 85, 7795-7799). This places FNDC5induction in a similar range to other known important regulators in thebrain.

In the study analyzing 30 days of free-wheel running exercise, Fndc5 andPgc1a was induced in the hippocampus but not in the rest of the brain(FIG. 1B) when taken as one unit. Therefore it is believed that Fndc5and Pgc1a were induced in relatively small numbers of neurons elsewhere,but that that change was not detectable because it is occurring in thebackground of little or no change in larger brain structures. Indeed,using a longer and more intense exercise regimen exercise protocol andmore detailed dissections, Steiner et al. reported an upregulation ofPgc1a expression in various other parts of the brain, in addition to thehippocampus (Steiner et al. (2011) J. Appl. Physiol. 111, 1066-1071).

In identifying how exercise is sensed by the brain (e.g., how the1α/FNDC5/BDNF pathway gets initiated in exercise), one obvious initiatorcould be increased neuronal activity in areas of the brain that areinvolved in spatial orientation, learning and memory, since BDNF geneexpression is well known to be stimulated by neural activity (West andGreenberg (2011) Cold Spring Harb. Perspect. Biol. 3, a005744).Increased sympathetic tone, namely higher norepinephrine levels (Garciaet al. (2003) Neurosci. 119, 721-732) and increased IGF-1 levels fromperiphery crossing the blood-brain-barrier have also been discussed asexercise-related inducers of BDNF (Ding et al. (2006) Neurosci. 140,823-833). However, because exercise is known to change the metabolicstate of the whole body, another important factor is believed to bechanges in the energy state or ox en levels within the brain, bothsignals to which PGC-1α gene expression is known to respond in othertissues (Arany et al. (2008) Nature 451, 1008-1012 and St-Pierre et al.(2006) Cell 127, 397-408). The experiments described herein linked theactivation of a metabolic regulator, PGC-1α, via FNDC5 to increased BDNFlevels in the neurons in response to exercise (FIG. 9) although otherimportant metabolic regulators exist, such as AMPK or PPARgamma, whichhave not been part of these studies.

FNDC5 in the periphery is cleaved and secreted as irisin and secretedirisin can cause the ‘browning’ of adipose tissues (Bostrom et al.(2012) Nature 481, 463-468; Shan et al. (2013) Faseb J. 27:1981-1989;and Wu et al. (2012) Cell 150, 366-376). It has been determined hereinthat peripheral delivery of FNDC5 with adenoviral vectors is sufficientto induce central expression of Bdnf and others genes with potentialneuroprotective functions or those involved in learning and memory. Thisimplies that a secreted, circulating form of FNDC5 has these effects onthese neurons and that it crosses the blood brain barrier. Thetherapeutic implications of this are large since it indicates that apolypeptide can provide neuroprotection in disease states or improvedcognition in aging populations.

EXAMPLE 10 FNDC5 Modulates Neuroprotective Signaling Pathways in Neurons

Primary cortical neurons were silenced at DIV6 with 1 uM TTX and 100 uMAPV overnight. The neurons were then stimulated at DIV7 using irisin (1ug/mL; Enzo; Product No. ADI-908-307-0010). FIG. 23 shows the results ofchanges in mRNA levels of genes of interest in the treated neuronsaccording to time after irisin stimulation and identifies that many ofthe analyzed genes have statistically significant changes with p<0.05from baseline. In a separate analysis, primary cortical neurons weresilenced at DIV6 with 1 uM TTX and 10 uM MK801 overnight. The neuronswere then stimulated at DIV6 using irisin (2 ug/mL; Phoenix; Product No.067-29A) and FIG. 24 shows the results of phosphorylated CREB responses.In still a separate analysis, primary cortical neurons were silenced atDIV6 with 1 uM TTX and 100 uM AP5 overnight. The neurons were thenstimulated at DIV6 using irisin (1 ug/mL; Enzo; Product No.ADI-908-307-0010). FIG. 25 shows the time course change of proteins ofinterest in the wild-type cells treated as described and analysesperformed in duplicate. In yet another separate analysis, primarycortical neurons were silenced at DIV7 with 1 uM TTX and 100 uM AP5overnight. The neurons were then stimulated at DIV8 using 1 ug/mL ofirisin from one of two vendors (i.e., either from Enzo, Product No.ADI-908-307-0010, or from Phoenix, Product No. 067-29A). FIG. 26 showsthe time course change of proteins of interest in the wild-type cellstreated as described and analyses performed in duplicate. These resultsindicate that FNDC5 modulates neuroprotective signaling pathways inneurons that are involved in improving cell survival, synapseplasticity, and learning and memory.

INCORPORATION BY REFERENCE

The contents of all references, patent applications, patents, andpublished patent applications, as well as the Figures and the SequenceListing, cited throughout this application are hereby incorporated byreference.

EQUIVALENTS

Those skilled, in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed:
 1. A method of increasing expression of brain-derivedneurotrophic factor (BDNF) by a cell comprising, contacting the cellwith an agent, wherein the agent is selected from the group consistingof an Fndc5 polypeptide or fragment thereof, a nucleic acid that encodesFndc5 or a fragment thereof and an enhancer of Fndc5 polypeptide and/ornucleic acid expression and/or activity, to thereby increase theexpression of BDNF by the cell.
 2. The method of claim 1, wherein thestep of contacting occurs in vivo, ex vivo, or in vitro.
 3. The methodof claim 1, wherein the cells are neurons.
 4. The method of claim 4,wherein the neurons are selected from the group consisting ofhippocampal neurons, cerebellar neurons, sciatic nerve neurons,dopaminergic neurons, and substantia nigra neurons.
 5. The method ofclaim 1, further comprising contacting the cell with an additional agentthat increases the expression of BDNF.
 6. A method for treating orpreventing a neurological disease or disorder in a subject comprisingthe step of administering to the subject an agent selected from thegroup consisting of an Fndc5 polypeptide or fragment thereof a nucleicacid that encodes Fndc5 or a fragment thereof, and an enhancer of Fndc5polypeptide and/or nucleic acid expression and/or activity, thatincreases BDNF expression or activity in the central or peripheralnervous system of the subject, such that the neurological, disease ordisorder is treated or prevented.
 7. The method of claim 6, wherein theagent is administered systemically.
 8. The method of claim 7, whereinthe systemic administration is intravenous or subcutaneous.
 9. Themethod of claim 6, wherein the agent is administered in apharmaceutically acceptable formulation.
 10. The method of claim 6,wherein the neurological disease or disorder would benefit fromdecreased neuronal cell death and/or increased neuronal survival,optionally wherein the neurological disease or disorder is selected fromthe group consisting of Alzheimer's disease, Parkinson's disease,Huntington's disease, Pick's disease, Kuf's disease, Lewy body disease,neurofibrillary tangles, Rosenthal fibers, Mallory's hyaline, seniledementia, myasthenia gravis, Gilles de la Tourette's syndrome, multiplesclerosis (MS), amyotrophic lateral sclerosis (ALS), progressivesupranuclear palsy (PSP), epilepsy, Creutzfeldt-Jakob disease,deafness-dytonia syndrome, Leigh syndrome, Leber hereditary opticneuropathy (LHON), parkinsonism, dystonia, motor neuron disease,neuropathy-ataxia and retinitis pimentosa (NARP), maternal inheritedLeigh syndrome (MILS), Friedreich ataxia, hereditary spastic paraplegia,Mohr-Tranebjaerg, syndrome, Wilson disease, sporatic Alzheimer'sdisease, sporadic amyotrophic lateral sclerosis, sporadic Parkinson'sdisease, autonomic function disorders, hypertension, sleep disorders,neuropsychiatric disorders, depression, schizophrenia, schizoaffectivedisorder, korsakoff's psychosis, mania, anxiety disorders, phobicdisorder, learning or memory disorders, amnesia or age-related memoryloss, attention deficit disorder, dysthymic disorder, major depressivedisorder, obsessive-compulsive disorder, psychoactive substance usedisorders, panic disorder, bipolar affective disorder, severe bipolaraffective (mood) disorder (BP-1), migraines, hyperactivity and movementdisorders.
 11. The method of claim 6, wherein the subject is a human.12. A method for assessing whether a subject is afflicted with aneurological disease or disorder or has a risk of developing aneurological disease or disorder comprising the steps of detecting theexpression of the Fndc5 gene or the expression or activity of Fndc5polypeptide in a sample of a subject, wherein a decrease in theexpression of the Fndc5 gene or a decrease in the expression or activityof the Fndc5 polypeptide compared to a control indicates the presence ofa neurological disease or disorder or the risk of developing aneurological disease or disorder in the subject.
 13. The method of claim12, wherein the sample is selected from the group consisting of wholeblood, serum, plasma, saliva, cerebrospinal fluid, spinal fluid, andneural tissue.
 14. The method of claim 12, wherein the expression of theFndc5 polypeptide or protein thereof is detected using a reagent whichspecifically binds with the protein.
 15. The method of claim 14, whereinthe reagent is selected from the group consisting of an antibody, anantibody derivative, and an antibody fragment.
 16. The method of claim12, wherein the expression of the Fndc5 gene is assessed by detectingthe presence in the sample of a transcribed polynucleotide or portionthereof.
 17. The method of claim 16, wherein the transcribedpolynucleotide is an mRNA or a cDNA.
 18. The method of claim 16, whereinthe step of detecting further comprises amplifying the transcribedpolynucleotide.
 19. The method of claim 16, wherein the level ofexpression of the marker in the sample is assessed by detecting thepresence in the sample of a transcribed polynucleotide which annealswith Fndc5 or anneals with a portion of an Fndc5 polynucleotide understringent hybridization conditions.
 20. The method of claim 12, whereinthe neurological disease or disorder would benefit from decreasedneuronal cell death and/or increased neuronal survival, optionallywherein the neurological disease or disorder is selected from the groupconsisting of Alzheimer's disease, Parkinson's disease, Huntington'sdisease, Pick's disease, Kuf's disease, Lewy body disease,neurofibrillary tangles, Rosenthal fibers, Mallory's hyaline, seniledementia, myasthenia gravis, Gilles de la Tourette's syndrome, multiplesclerosis (MS), amyotrophic lateral sclerosis (ALS), progressivesupranuclear palsy (PST), epilepsy, Creutzfeldt-Jakob disease,deafness-dytonia syndrome, Leigh syndrome, Leber hereditary opticneuropathy (LHON), parkinsonism, dystonia, motor neuron disease,neuropathy-ataxia and retinitis pimentosa (NARP), maternal inheritedLeigh syndrome (MILS), Friedreich ataxia, hereditary spastic paraplegia,Mohr-Tranebjaerg syndrome, Wilson disease, sporatic Alzheimer's disease,sporadic amyotrophic lateral sclerosis, sporadic Parkinson's disease,autonomic function disorders, hypertension, sleep disorders,neuropsychiatric disorders, depression, schizophrenia, schizoaffectivedisorder, korsakoff's psychosis, mania, anxiety disorders, phobicdisorder, learning or memory disorders, amnesia or age-related memoryloss, attention deficit disorder, dysthymic disorder, major depressivedisorder, obsessive-compulsive disorder, psychoactive substance usedisorders, panic disorder, bipolar affective disorder, severe bipolaraffective (mood) disorder (BP-1), migraines, hyperactivity and movementdisorders.
 21. The method of claim 12, wherein the subject is a human.22. A method for assessing the efficacy of an agent that treats orprevents a neurological disease or disorder in a subject, comprising: a)detecting in a subject sample at a first point in time BDNF polypeptideor nucleic acid expression and/or activity in the central and/orperipheral nervous system; b) repeating step a) during at least onesubsequent point in time after administration of the agent, wherein theagent is selected from the group consisting of an Fndc5 polypeptide orfragment thereof, a nucleic acid that encodes Fndc5 or a fragmentthereof, and an enhancer of Fndc5 polypeptide and/or nucleic acidexpression and/or activity; and c) comparing the expression and/oractivity detected in steps a) and b), wherein a significantly increasedBDNF polypeptide or nucleic acid expression and/or activity in the firstsubject sample relative to at least one subsequent subject sample,indicates that the agent treats or prevents the neurological disease ordisorder in the subject.
 23. The method of claim 22, wherein the firstand/or at least one subsequent sample is selected from the groupconsisting of whole blood, serum, plasma, saliva, cerebrospinal fluid,spinal fluid, and neural tissue.
 24. The method of claim 22, whereinbetween the first point in time and the subsequent point in time, thesubject has undergone treatment, completed treatment, and/or is inremission for the neurological disease or disorder.
 25. The method ofclaim 22, wherein the first and/or at least one subsequent sample isselected from the group consisting of ex vivo and in vivo samples. 26.The method of claim 22, wherein the first and/or at least one subsequentsample is obtained from an animal model of the neurological disease ordisorder.
 27. The method of claim 22, wherein the first and/or at leastone subsequent sample is a portion of a single sample or pooled samplesobtained from the subject.
 28. The method of claim 22, wherein theexpression of the BDNF polypeptide is detected using a reagent whichspecifically binds with the protein.
 29. The method of claim 28, whereinthe reagent is selected from the group consisting of an antibody, anantibody derivative, and an antibody fragment.
 30. The method of claim22, wherein the expression of the BDNF nucleic acid is assessed bydetecting the presence in the sample of a transcribed polynucleotide orportion thereof.
 31. The method of claim 30, wherein the transcribedpolynucleotide is an mRNA or a cDNA.
 32. The method of claim 30, whereinthe step of detecting further comprises amplifying the transcribedpolynucleotide.
 33. The method of claim 30, wherein the level ofexpression of the marker in the sample is assessed by detecting thepresence in the sample of a transcribed polynucleotide which annealswith BDNF or anneals with a portion of a BDNF polynucleotide understringent hybridization conditions.
 34. A cell-based assay for screeningfor agents that modulate the ability of the cell to increase BDNFexpression comprising contacting the cell with a test agent selectedfrom the group consisting of an Fndc5 polypeptide or fragment thereof, anucleic acid that encodes Fndc5 or a fragment thereof, and an enhancerof Fndc5 polypeptide and/or nucleic acid expression and/or activity, anddetermining the ability of the test agent to increase BDNF expression bythe cell.
 35. The assay of claim 34, wherein the step of contactingoccurs in vivo, ex vivo, or in vitro.
 36. The assay of claim 34, whereinthe cells are neurons.
 37. The assay of claim 36, wherein the neuronsare selected from the group consisting of hippocampal neurons,cerebellar neurons, sciatic nerve neurons, dopaminergic neurons, andsubstantia nigra neurons.
 38. The method or assay of any precedingclaims, wherein the Fndc5 polypeptide is selected from the group ofpolypeptides consisting of: a) a polypeptide encoded by a nucleic acidmolecule comprising a nucleotide sequence encoding a fragment of theFNDC5 polypeptide of SEQ ID NO: 2, wherein said fragment lacks theC-terminal domain sequence of said FNDC5 polypeptide and wherein saidpolypeptide has one or more of the biological activities of said FNCD5polypeptide; b) an isolated polypeptide encoded by a nucleic acidmolecule comprising a nucleotide sequence encoding an amino acidsequence that is at least 70% identical to the amino acid sequence ofresidues 73-140 of the FNDC5 polypeptide of SEQ ID NO:2, wherein saidpolypeptide does not encode the C-terminal domain sequence of said FNDC5polypeptide, and wherein said polypeptide has one or more of thebiological activities of said FNCD5 polypeptide; c) a polypeptide whichis a fragment of the FNDC5 polypeptide of SEQ ID NO: 2, which fragmentis optionally fused to one or more heterologous polypeptides at itsN-terminus and/or C-terminus, wherein said fragment consists of asequence of amino acids in between residues 1 and 150 of SEQ ID NO: 2,and wherein said fragment has one or more of the biological activitiesof said FNDC5 polypeptide; and d) a polypeptide which is a fragment ofthe FNDC5 polypeptide of SEQ ID NO: 4, 6 or 8, wherein said fragment isoptionally fused to one or more heterologous polypeptides at itsN-terminus and/or C-terminus, and wherein said fragment has one or inureof the biological activities of said FNCD5 polypeptide.
 39. The methodor assay of any preceding claim, wherein the Fndc5 polypeptide isselected from the group of polypeptides consisting of: a) an isolatedpolypeptide fragment of an Fndc5 protein comprising at least onefibronectin domain and is not full-length Fndc5; b) an isolatedpolypeptide fragment of an Fndc5 protein comprising at least onefibronectin domain and which lacks one or more functional domain(s)selected from the group consisting of signal peptide, hydrophobic, andC-terminal domains; c) an isolated polypeptide comprising an amino acidsequence that is at least 70% identity to the amino acid sequencecomprising residues 73-140 of SEQ ID NO:2, residues 30-140 of SEQ IDNO:2 or residues 29-140 of SEQ ID NO:2 and which lacks one or morefunctional domain(s) of an Fndc5 protein selected from the groupconsisting of signal peptide, hydrophobic, and C-terminal domains; d) anisolated polypeptide comprising an amino acid sequence that is at least70% identity to the amino acid sequence comprising residues 73-140 ofSEQ ID NO:2, residues 30-140 of SEQ ID NO:2 or residues 29-140 of SEQ IDNO:2 and which is less than 195 amino acids in length; e) an isolatedpolypeptide consisting essentially of an amino acid sequence that is atleast 70% identity to the amino acid sequence comprising residues 73-140of SEQ ID NO:2, residues 30-140 of SEQ ID NO:2 or residues 29-140 of SEQID NO:2; f) an isolated polypeptide fragment of SEQ ID NO:2 comprisingresidues 73-140 of SEQ ID NO:2, residues 30-140 of SEQ ID NO:2 orresidues 29-140 of SEQ ID NO:2 and which is not full-length; g) anisolated polypeptide fragment of SEQ ID NO: 12 consisting essentially ofresidues 73-140 of SEQ ID NO:2, residues 30-140 of SEQ ID NO:2 orresidues 29-140 of SEQ ID NO:2; h) an isolated polypeptide which isencoded by a nucleic acid molecule comprising a nucleotide sequenceencoding at least one fibronectin domain of an Fndc5 protein and doesnot encode full-length Fndc5; i) an isolated polypeptide fragment of anFndc5 protein which is encoded by a nucleic acid molecule comprising anucleotide sequence encoding at least one fibronectin domain and whichdoes not encode one or more functional domain(s) selected from the groupconsisting of signal peptide, hydrophobic, and C-terminal domains; j) anisolated polypeptide which is encoded by a nucleic acid moleculecomprising a nucleotide sequence encoding an amino acid sequence that isat least 70% identical to the amino acid sequence of residues 73-140 ofSEQ ID NO:12, residues 30-140 of SEQ ID NO:2 or residues 29-140 of SEQID NO:12 and which does not encode one or more functional domain(s) ofan Fndc5 protein selected from the group consisting of signal peptide,hydrophobic, and C-terminal domains; k) an isolated polypeptide which isencoded by a nucleic acid molecule comprising a nucleotide sequenceencoding an amino acid sequence that is at least 70% identical to theamino acid sequence of residues 73-140 of SEQ ID NO:12, residues 30-140of SEQ ID NO:2 or residues 29-140 of SEQ ID 4O:2 and which is less than630 nucleotides in length; l) an isolated polypeptide which is encodedby a nucleic acid molecule consisting essentially of a nucleotidesequence encoding an amino acid sequence having at least 70% identity tothe amino acid sequence of residues 73-140 of SEQ ID NO:2, residues30-140 of SEQ ID NO:2 or residues 29-140 of SEQ ID NO:2; m) an isolatedpolypeptide which is encoded by a nucleic acid molecule comprising anucleotide sequence encoding an amino acid sequence that is at least 70%identical to the amino acid sequence of residues 73-140 of SEQ IDNO:2residues 30-140 of SEQ ID NO:2 or residues 29-140 of SEQ ID NO:2 andwhich does not encode the full-length amino acid sequence of SEQ IDNO:2: n) an isolated polypeptide which is encoded by a nucleic acidmolecule comprising a nucleotide sequence encoding the amino acidsequence of residues 73-140 of SEQ ID NO:2, residues 30-140 of SEQ IDNO:2 or residues 29-140 of SEQ ID NO:2 and which does not encode thefull-length amino acid sequence of SEQ ID NO:2; o) an isolatedpolypeptide which is encoded by a nucleic acid molecule consistingessentially of a nucleotide sequence encoding the amino acid sequence ofresidues 73-140 of SEQ ID NO:2, residues 30-140 of SEQ ID NO:2 orresidues 29-140 of SEQ ID NO:2: p) an isolated polypeptide which isencoded by a nucleic acid molecule comprising a nucleotide sequencewhich is at least 70% identical to the nucleotide sequence ofnucleotides 217-420 of SEQ ID NO:1, SEQ ID NO:15, nucleotides 88-420 ofSEQ ID NO:1, or nucleotides 85-420 of SEQ ID NO:1 and which does notencode one or more functional domain(s) of an Fndc5 protein selectedfrom the group consisting of signal peptide, hydrophobic, and C-terminaldomains; and q) an isolated polypeptide which is encoded by a nucleicacid molecule consisting essentially of a nucleotide sequence which isat least 70% identical to the nucleotide sequence of nucleotides 217-420of SEQ ID NO:1, SEQ ID NO:15, nucleotides 88-420 of SEQ ID NO:1, ornucleotides 85-420 of SEQ NO:1.
 40. The method or assay of any precedingclaim, wherein the one or more of the biological activities of FNDC5polypeptide is selected from the group consisting of 1) increasing BDNFexpression in the central and/or peripheral nervous system; 2)increasing activity-induced immediate-early gene expression in neurons;3) increasing neuronal survival; 4) decreasing neurological lesionformation; 5) increasing neurite outgrowth; 6) increasingsynaptogenesis; 7) increasing synaptic plasticity; 8) decreasingneuronal mitochondrial dysfunction, 9) increasing dendriticarborization; 10) increasing neuronal differentiation; and 11)increasing neuronal migration.
 41. The method or assay of any precedingclaim, wherein said fragment or encoded amino acid sequence is more than65 amino acids in length and/or less than 135 amino acids in length. 42.The method or assay of any preceding claim, wherein said polypeptide isbetween 70 and 125 amino acids in length or is less than 195 amino acidsin length.
 43. The method or assay of any preceding claim, wherein saidpolypeptide is a fragment of SEQ ID NO: 2 which consists of about aminoacids 30 to 140 or 73-140 of SEQ ID NO: 2, wherein said fragment isoptionally fused to one or more heterologous polypeptides at itsN-terminus and/or C-terminus.
 44. The method or assay of any precedingclaim, wherein said polypeptide comprises a fibronectin domain.
 45. Themethod or assay of any preceding claim, wherein said polypeptide isglycosylated or pegylated, optionally wherein at least one glycosylatedamino acid residue corresponds to asparagine at position 36 and/or theasparagine at position 81 of SEQ ID NO:
 2. 46. The method or assay ofany preceding claim, wherein said polypeptide comprises an amino acidsequence that is heterologous to said FNDC5 polypeptide.
 47. The methodor assay of claim 46, wherein said heterologous ammo acid sequence is anFc domain, an IgG1 Fc domain, an IgG2 Fc domain, an IgG3 Fc domain, andIgG4 Fc domain, a dimerization domain, an oligomerization domain, anagent that promotes plasma solubility, albumin, a signal peptide, apeptide tag, a 6-His tag, a thioredoxin tag, a hemaglutinin tag, a GSTtag, or an OmpA signal sequence tag.
 48. The method or assay of anypreceding claim, wherein said polypeptide can cross the blood-brainbarrier.
 49. The method or assay of any preceding claim, wherein saidFndc5 nucleic acid encodes a polypeptide of claim 38-48.
 50. The methodor assay of any preceding claim, wherein said Fndc5 nucleic acid isselected from the group consisting of: a) a nucleic acid moleculecomprising a nucleotide sequence encoding a fragment of the FNDC5polypeptide of SEQ ID NO: 2, wherein said fragment lacks the C-terminaldomain sequence of said FNDC5 polypeptide, and wherein said fragment hasone or more of the biological activities of said FNCD5 polypeptide; b) anucleic acid molecule which encodes a polypeptide comprising an aminoacid sequence having at least 70% identity to the amino acid sequence ofresidues 73-140 of the FNDC5 polypeptide of SEQ ID NO:2, wherein saidpolypeptide does not encode the C-terminal domain sequence of said FNDC5polypeptide, and wherein said polypeptide has one or more of thebiological activities of said FNCD5 polypeptide; and c) a nucleic acidmolecule which encodes a fibronectin domain of the FNCD5 polypeptide ofSEQ ID NO: 2 but which does not encode the full length sequence of SEQID NO:
 2. 51. The method or assay of any preceding claim, wherein saidFndc5 nucleic acid is selected from the group consisting of: a) anisolated nucleic acid molecule which encodes at least one fibronectindomain of an Fndc5 protein and which does not encode full-length Fndc5;b) an isolated nucleic acid molecule which encodes at least onefibronectin domain of an Fndc5 protein and which does not encode one ormore functional domain(s) of an Fndc5 protein selected from the groupconsisting of signal peptide, hydrophobic, and C-terminal domains; c) anisolated nucleic acid molecule which encodes a polypeptide comprising anamino acid sequence having at least 70% identity to the 88-amino acidsequence of residues 73-140 of SEQ ID NO:2, residues 30-140 of SEQ IDNO:2 or residues 29-140 of SEQ ID NO:2 and which does not encode one ormore functional domain(s) of an Fndc5 protein selected from the groupconsisting of signal peptide, hydrophobic, and C-terminal domains; d) anisolated nucleic acid molecule which encodes a polypeptide comprising anamino acid sequence having at least 70% identity to the amino acidsequence of residues 73-140 of SEQ ID NO:2, residues 30-140 of SEQ IDNO:2 or residues 29-140 of SEQ ID NO:2 and which is less than 630nucleotides in length; e) an isolated nucleic acid molecule whichencodes a polypeptide consisting essentially of an amino acid sequencehaving at least 70% identity to the amino acid sequence of residues73-140 of SEQ ID NO:2, residues 30-140 of SEQ ID NO:2 or residues 29-140of SEQ ID NO:2; f) an isolated nucleic acid molecule which encodes apolypeptide comprising an amino acid sequence having at least 70%identity to the amino acid sequence of residues 73-140 of SEQ ID NO:2,residues 30-140 of SEQ ID NO:2 or residues 29-140 of SEQ ID NO:2 andwhich does not encode the full-length amino acid sequence of SEQ IDNO:2; g) isolated nucleic acid molecule which encodes a polypeptidecomprising the amino acid sequence of residues 73-140 of SEQ ID NO:2,residues 30-140 of SEQ ID NO:2 or residues 29-140 of SEQ ID NO:2 andwhich does not encode the full-length amino acid sequence of SEQ IDNO:12; h) an isolated nucleic acid molecule which encodes a polypeptideconsisting essentially of the amino acid sequence of residues 73-140 ofSEQ ID NO:2, residues 30-140 of SEQ ID NO:2 or residues 29-140 of SEQ IDNO:2; i) an isolated nucleic acid molecule comprising a nucleotidesequence which is at least 70% identical to the nucleotide sequence ofnucleotides 217-420 of SEQ ID NO:1, SEQ ID NO:15, nucleotides 88-420 ofSEQ ID NO:1, or nucleotides 85-420 of SEQ ID NO:1 and which does notencode one or more functional domain(s) of an Fndc5 protein selectedfrom the group consisting of signal peptide, hydrophobic, and C-terminaldomains; and j) an isolated nucleic acid molecule consisting essentiallyof a nucleotide sequence which is at least 70% identical to thenucleotide sequence of nucleotides 217-420 of SEQ ID NO:1, SEQ ID NO:15,nucleotides 88-420 of SEQ ID NO:1, or nucleotides 85-420 of SEQ NO:1.